Heterodimeric proteins

ABSTRACT

The present invention describes novel immunoglobulin compositions that co-engage at least two antigens, e.g. a first and second antigen, or, as outlined herein, three or four antigens can be bound, in some of the scaffold formats described herein. First and second antigens of the invention are herein referred to as antigen-1 and antigen-2 respectively (or antigen-3 and antigen-4, if applicable. As outlined herein, a number of different formats can be used, with some scaffolds relying combinations of monovalent and bivalent bindings.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 61/794,695, filed Mar. 15, 2013, which is incorporated herein byreference in its entirety.

INCORPORATION OF RELATED APPLICATIONS

The following applications are incorporated by reference in theirentirety, U.S. Ser. Nos. 61/302,707, 61/732,813, 61/598,686, 61/441,552,13/648,951, 12/875,015; 61/311,472; 61/450,457; 61/545,498; 61/239,316;13/568,028; 61/515,745; 61/785,241; 61/785,265; 61/752,349; 61/764,954;61/780,310; 61/780,334; 13/194,904; 61/368,969; 61/391,509; 61/391,515;61/439,263; 61/593,846; 61/368,962.

BACKGROUND OF THE INVENTION

Antibody-based therapeutics have been used successfully to treat avariety of diseases, including cancer and autoimmune/inflammatorydisorders. Yet improvements to this class of drugs are still needed,particularly with respect to enhancing their clinical efficacy. Oneavenue being explored is the engineering of additional and novel antigenbinding sites into antibody-based drugs such that a singleimmunoglobulin molecule co-engages two different antigens. Suchnon-native or alternate antibody formats that engage two differentantigens are often referred to as bispecifics. Because the considerablediversity of the antibody variable region (Fv) makes it possible toproduce an Fv that recognizes virtually any molecule, the typicalapproach to bispecific generation is the introduction of new variableregions into the antibody.

A number of alternate antibody formats have been explored for bispecifictargeting (Chames & Baty, 2009, mAbs 1[6]:1-9; Holliger & Hudson, 2005,Nature Biotechnology 23[9]:1126-1136; Kontermann, mAbs 4(2):182 (2012),all of which are expressly incorporated herein by reference). Initially,bispecific antibodies were made by fusing two cell lines that eachproduced a single monoclonal antibody (Milstein et al., 1983, Nature305:537-540). Although the resulting hybrid hybridoma or quadroma didproduce bispecific antibodies, they were only a minor population, andextensive purification was required to isolate the desired antibody. Anengineering solution to this was the use of antibody fragments to makebispecifics. Because such fragments lack the complex quaternarystructure of a full length antibody, variable light and heavy chains canbe linked in single genetic constructs. Antibody fragments of manydifferent forms have been generated, including diabodies, single chaindiabodies, tandem scFv's, and Fab₂ bispecifics (Chames & Baty, 2009,mAbs 1[6]:1-9; Holliger & Hudson, 2005, Nature Biotechnology23[9]:1126-1136; expressly incorporated herein by reference). Whilethese formats can be expressed at high levels in bacteria and may havefavorable penetration benefits due to their small size, they clearrapidly in vivo and can present manufacturing obstacles related to theirproduction and stability. A principal cause of these drawbacks is thatantibody fragments typically lack the constant region of the antibodywith its associated functional properties, including larger size, highstability, and binding to various Fc receptors and ligands that maintainlong half-life in serum (i.e. the neonatal Fc receptor FcRn) or serve asbinding sites for purification (i.e. protein A and protein G).

More recent work has attempted to address the shortcomings offragment-based bispecifics by engineering dual binding into full lengthantibody-like formats (Wu et al., 2007, Nature Biotechnology25[11]:1290-1297; U.S. Ser. No. 12/477,711; Michaelson et al., 2009,mAbs 1[2]:128-141; PCT/US2008/074693; Zuo et al., 2000, ProteinEngineering 13[5]:361-367; U.S. Ser. No. 9/865,198; Shen et al., 2006, JBiol Chem 281[16]:10706-10714; Lu et al., 2005, J Biol Chem280[20]:19665-19672; PCT/US2005/025472; expressly incorporated herein byreference). These formats overcome some of the obstacles of the antibodyfragment bispecifics, principally because they contain an Fc region. Onesignificant drawback of these formats is that, because they build newantigen binding sites on top of the homodimeric constant chains, bindingto the new antigen is always bivalent.

For many antigens that are attractive as co-targets in a therapeuticbispecific format, the desired binding is monovalent rather thanbivalent. For many immune receptors, cellular activation is accomplishedby cross-linking of a monovalent binding interaction. The mechanism ofcross-linking is typically mediated by antibody/antigen immunecomplexes, or via effector cell to target cell engagement. For example,the low affinity Fc gamma receptors (FcγRs) such as FcγRIIa, FcγRIIb,and FcγRIIIa bind monovalently to the antibody Fc region. Monovalentbinding does not activate cells expressing these FcγRs; however, uponimmune complexation or cell-to-cell contact, receptors are cross-linkedand clustered on the cell surface, leading to activation. For receptorsresponsible for mediating cellular killing, for example FcγRIIIa onnatural killer (NK) cells, receptor cross-linking and cellularactivation occurs when the effector cell engages the target cell in ahighly avid format (Bowles & Weiner, 2005, J Immunol Methods 304:88-99,expressly incorporated by reference). Similarly, on B cells theinhibitory receptor FcγRIIb downregulates B cell activation only when itengages into an immune complex with the cell surface B-cell receptor(BCR), a mechanism that is mediated by immune complexation of solubleIgG's with the same antigen that is recognized by the BCR (Heyman 2003,Immunol Lett 88[2]:157-161; Smith and Clatworthy, 2010, Nature ReviewsImmunology 10:328-343; expressly incorporated by reference). As anotherexample, CD3 activation of T-cells occurs only when its associatedT-cell receptor (TCR) engages antigen-loaded MHC on antigen presentingcells in a highly avid cell-to-cell synapse (Kuhns et al., 2006,Immunity 24:133-139). Indeed nonspecific bivalent cross-linking of CD3using an anti-CD3 antibody elicits a cytokine storm and toxicity(Perruche et al., 2009, J Immunol 183 [2]:953-61; Chatenoud & Bluestone,2007, Nature Reviews Immunology 7:622-632; expressly incorporated byreference). Thus for practical clinical use, the preferred mode of CD3co-engagement for redirected killing of targets cells is monovalentbinding that results in activation only upon engagement with theco-engaged target.

Thus while bispecifics generated from antibody fragments sufferbiophysical and pharmacokinetic hurdles, a drawback of those built withfull length antibody-like formats is that they engage co-target antigensmultivalently in the absence of the primary target antigen, leading tononspecific activation and potentially toxicity. The present inventionsolves this problem by introducing a novel set of bispecific formatsthat enable the multivalent co-engagement of distinct target antigens.

BRIEF SUMMARY OF THE INVENTION

The present invention describes novel immunoglobulin compositions thatco-engage at least two antigens, e.g. a first and second antigen, or, asoutlined herein, three or four antigens can be bound, in some of thescaffold formats described herein. First and second antigens of theinvention are herein referred to as antigen-1 and antigen-2 respectively(or antigen-3 and antigen-4, if applicable. As outlined herein, a numberof different formats can be used, with some scaffolds relyingcombinations of monovalent and bivalent bindings.

In preferred embodiments of the invention, the antigen binding regionsof the immunoglobulin are antibody variable regions. In theseembodiments, binding to antigens is mediated by variable regions, alsoreferred to as Fv regions, each comprising a VH domain and a VL domain.The Fv region that binds antigen-1 is referred to as Fv-1, while the Fvregion that binds antigen-2 is referred to as Fv-2, etc. However, asoutlined herein, ligands can also be used in the “Fc fusion” constructsoutlined herein.

The present invention describes methods for generating the novelcompositions of the invention. The present invention describespurification methods for the immunogloublins herein, particularlymethods for separating heterodimeric and homodimeric protein species.Also described are methods of testing the immunoglobulins herein,including in vitro and in vitro experiments.

The present invention provides isolated nucleic acids encoding the novelimmunoglobulin compositions described herein. The present inventionprovides vectors comprising said nucleic acids, optionally, operablylinked to control sequences. The present invention provides host cellscontaining the vectors, and methods for producing and optionallyrecovering the immunoglobulin compositions.

The present invention provides compositions comprising immunoglobulinpolypeptides described herein, and a physiologically or pharmaceuticallyacceptable carrier or diluent.

The present invention contemplates therapeutic and diagnostic uses forthe immunoglobulin polypeptides disclosed herein.

Thus, in one aspect, the present invention provides compositionscomprising a heterodimer protein comprising a first monomer comprising afirst variant heavy chain constant region and a first fusion partner;and a second monomer comprising: a second variant heavy chain constantregion and a second fusion partner. In some cases the heterodimericproteins are constructed such that the isoelectric points (pIs) of thefirst and second variant heavy chain constant regions are at least 0.5logs apart. In additional cases, the Fc region of the first and secondconstant regions comprise a set of amino acid substitutions from FIG.79. In additional cases, the Fc region of said first and second constantregions comprise a set of amino acid substitutions from FIG. 80. Infurther cases, the Fc region of the first and second constant regionscomprise a set of amino acid substitutions from FIG. 82.

In further aspects, the heterodimeric protein compositions of theinvention has a structure selected from the group consisting of thestructures in FIGS. 78A-78N and 78P-78S.

In an addition aspect, any of the heterodimeric proteins, particularlyheterodimeric antibodies, has a first monomer comprising at these the pIsubstitutions ISO(−):I199T/N203D/K274Q/R355Q/N384S/K392NN397M/Q419E/DEL44 and monomer 2comprises pI substitutions ISO(+RR): Q196K/I199T/P217R/P228R/N276K.

In further aspects, the heterodimeric proteins of the invention can havea third fusion partner. In some aspects, the heterodimeric proteins ofthe invention can have a fourth fusion partner.

In further aspects, the fusion partners are independently selected fromthe group consisting of an immunoglobulin component, a peptide, acytokine, a chemokine, an immune receptor and a blood factor. Theimmunoglobulin component can be selected from the group consisting ofFab, VH, VL, scFv, scFv2, dAb. In some cases, two, three or four of thefusion partners are immunoglobulin components, in particular, scFv andFab components find particular use as fusion partners. In some cases,the fusion partner cytokine is selected from the group consisting ofIL-2, IL-10, IL-12 and GM-CSF. In some cases, the fusion partnerchemokine is selected from the group consisting of RANTES, CXCL9, CXCL10and CXCL12. In some cases, the fusion partner immune receptor isselected from the group consisting of CTLA-4, TNFRI, TNFRII, a TNFSFprotein, and TNFRSF. In some cases, the fusion partner blood factor isselected from the group consisting of Factor VII, Factor VIII and FactorIX. Any and all of these fusion partners may be independently andoptionally combined with any other.

In an additional aspect, at least one Fc domain of one monomer heavychain comprises an amino acid variant selected from the group consistingof 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 243L,298A and 299T. In these cases, the heterodimeric protein can havealtered binding to FcγR receptors, particularly increased binding toFcγRIIb and/or FcγRIIIa. IN some cases both monomers comprise the Fcvariants.

In a further aspect, at least one Fc domain of one monomer heavy chaincomprises an amino acid variant selected from the group consisting of434A, 434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I orV/434S, 436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. In thesecases, the heterodimeric protein can have altered binding to FcRnreceptors, particularly increased binding. In some cases both monomerscomprise the Fc variants.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. Amino acid sequences of wild-type constant regions used in theinvention.

FIG. 2A-2C. Engineering of heavy chain CH1 domains. List of CH1 residuesfor the four IgG isotypes, fraction exposed, and examples ofsubstitutions that can be made to lower pI. Numbering is according tothe EU index.

FIG. 3A-3C. Engineering of light chain CK domains. List of CK residues,fraction exposed, and substitutions that can be made to lower pI.Numbering is according to the EU index.

FIG. 4. Amino acid sequences of pI engineered constant regionsIgG1-CH1-pI(6) and CK-pI(6).

FIG. 5. Amino acid sequences of wild-type anti-VEGF VH and VL variableregions used in the invention.

FIG. 6. Amino acid sequences of the heavy and light chains of pIengineered anti-VEGF antibody XENP9493 IgG1-CH1-pI(6)-CK-pI(6) used inthe invention.

FIG. 7. Structure of an antibody Fab domain showing the locations of pIlowering mutations in XENP9493 IgG1-CH1-pI(6)-CK-pI(6).

FIG. 8. Analysis of pI engineered anti-VEGF variants on an AgilentBioanalyzer showing high purity.

FIG. 9A-9C. Analysis of pI engineered anti-VEGF variants on SEC showinghigh purity.

FIG. 10. Analysis of pI engineered anti-VEGF variants on an IEF gelshowing variants have altered pI.

FIG. 11A-11B. Binding analysis (Biacore) of bevacizumab and pIengineered anti-VEGF binding to VEGF.

FIG. 12. DSC analysis of CH1 and CK pI engineered anti-VEGF showing highthermostability.

FIG. 13. PK of bevacizumab variants in huFcRn mice. The 9493 variantwith pI-engineered CH1 and CK domains extends half-life in vivo.

FIG. 14. PK of a native IgG1 version of bevacizumab in four separate invivo studies in huFcRn mice. The average IgG1 half-life was 3.2 days.

FIG. 15. PK of a native IgG2 version of bevacizumab in huFcRn mice.

FIG. 16. Correlation between half-life and isoelectric point (pI) ofantibody variants with different constant chains.

FIG. 17A-17D. Amino acid sequence alignment of the IgG subclasses.Residues with a bounded box illustrate isotypic differences between theIgG's. Residues which contribute to a higher pI (K, R, and H) or lowerpI (D and E) are highlighted in bold. Designed substitutions that eitherlower the pI, or extend an epitope are shown in gray.

FIG. 18. Amino acid sequence of the CK and Cλ light constant chains.Residues which contribute to a higher pI (K, R, and H) or lower pI (Dand E) are highlighted in bold. Preferred positions that can be modifiedto lower the pI are shown in gray.

FIG. 19A-19B. Amino acid sequences of pI-engineered variant heavychains.

FIG. 20. Amino acid sequences of pI-engineered variant light chains.

FIG. 21. PK results of pI-engineered variant bevacizumab antibodies inhuFcRn mice.

FIG. 22. PK results of variants that combine pI-engineered modificationswith Fc modifications that enhance binding to FcRn.

FIG. 23. Correlation between half-life and isoelectric point (pI) ofnative bevacizumab antibodies, pI-engineered variant versions withreduced pI, and native and pI-engineered versions that incorporate Fcmodifications that improve binding to human FcRn.

FIG. 24A-24C Amino acid sequence alignment of novel isotype IgG-pI-Iso3with the IgG subclasses. Blue indicates a match between pI-iso3 andresidues in the four native IgG's IgG1, IgG2, IgG3, and IgG4. Residueswith a bounded box illustrate IgG isotypic differences that have beenincorporated into IgG-pI-Iso3 that reduce pI.

FIG. 25. Differences between IgG1 and IgG-pI-Iso3 in the hinge and Fcregion.

FIG. 26. Differences between IgG1 and IgG-pI-Iso3 in the CH1 region.

FIG. 27. Amino acid illustration of the CK-pI(4) variant. Red indicateslysine to glutamic acid charge substitutions relative to the native CKlight constant chain.

FIG. 28A-28D Amino acid sequences of pI-engineered heavy and lightconstant chains.

FIG. 29. Analysis of basic residues in the antibody Fc region showingfraction exposed and the calculated energy for substitution to Glunormalized against the energy of the WT residue. Basic residues with ahigh fraction exposed and a favorable delta E for substitution to Gluare targets for charge swap mutations to lower pI.

FIG. 30. Plot showing the effect of charge swap mutations on antibodypI. As the pI gets lower the change in pI per charge swap decreases.

FIG. 31. PK results of pI-engineered isotypic variant bevacizumabantibodies (IgG-pI-Iso3) and combinations with substitution N434S inhuFcRn mice.

FIG. 32. PK results of pI-engineered isotypic variant bevacizumabantibodies and combinations with substitution N434S in huFcRn mice.

FIG. 33. Scatter plot of PK results of pI-engineered isotypic variantbevacizumab antibodies and combinations with substitution N434S inhuFcRn mice. Each point represents a single mouse from the study. Itshould be noted that the 428L substitution can also be added to each ofthese pI antibodies.

FIG. 34. Plot showing correlation between pI engineered variant pI andhalf-life (t½).

FIG. 35. Structural alignment of CK and C-lambda domains.

FIG. 36. Literature pIs of the 20 amino acids. It should be noted thatthe listed pIs are calculated as free amino acids; the actual pI of anyside chain in the context of a protein is different, and thus this listis used to show pI trends and not absolute numbers for the purposes ofthe invention.

FIG. 37A-37F. Data table of exemplary pI-engineered variants listing:

XenP# the internal reference number Name (HC) heavy chain sequencedesignation SEQ ID NO (HC) corresponding SEQ ID NO of the heavy chainsequence Name (LC) light chain sequence designation SEQ ID NO (LC)corresponding SEQ ID NO of the light chain sequence Calc. pI calculatedpI value for the entire antibody sequence, including heavy and lightchain Fv + constant domains, with the Fv of bevacizumab and the constantdomains as defined in the table #KR number of Lys or Arg residues inIgG1 with the Fv of bevacizumab and the constant domains as defined inthe table Delta KR (vs. change in the number of Lys or Arg residuesrelative WT) to IgG1 wild-type sequence of bevacizumab #DE number of Aspor Glu residues in IgG1 with the Fv of bevacizumab and the constantdomains as defined in the table Delta DE (vs. change in the number ofAsp or Glu acid residues WT) relative to IgG1 wild-type sequence ofbevacizumab Charge state derived from the total number of Lys and Argminus the total number of Asp and Glu residues, assuming a pH of 7 # HCMutations number of mutations in the heavy chain constant vs IgG1 domainas compared to IgG1 # LC Mutations number of mutations in the lightchain constant vs IgG1 domain as compared to IgG1 Total # of totalnumber of mutations in the heavy chain and light Mutations chainconstant domains as compared to IgG1

It should be noted that FIG. 20 has SEQ ID NO:s that are associated withthe sequence listing filed in U.S. Ser. No. 13/648,951, and are herebyexpressly incorporated by reference.

FIG. 38. Outline of method of purifying a desired heterodimeric antibodyspecies from a mixture of contaminating homodimer species by engineeringto modify isoelectric points of individual chains. As will beappreciated by those in the art, while the schematic is shown for a“standard” bispecific antibody format, the method is the same for othermultispecific heterodimers relying on pI variants for purification; seefor example

FIG. 39A-39E. Sequences of pI engineered variants, includingheterodimeric and bispecific constructs.

FIG. 40. IEF gel showing purification of the heterodimer species of thepI engineered variant XENP10653 from the homodimer species by anionexchange chromatography. As can be seen from lane 3, the desiredheterodimer is obtained in high purity.

FIG. 41. Outline of method of purifying a desired heterodimericbispecific Mab-Fv from a mixture of contaminating homodimer species byengineering to modify isoelectric points of individual chains.

FIG. 42. Outline of method of purifying a desired heterodimericbispecific scFv-Fc from a mixture of contaminating homodimer species byengineering to modify isoelectric points of individual chains.

FIG. 43A-43E. List of heavy chain and light chain residues for humanIgG1 and percent exposed surface area. Numbering is according to the EUindex.

FIG. 44A-44G. Examples of acidic substitutions that can be made in theheavy chain to facilitate easy purification of a heterodimeric species.Calculated pI in the context of bevacizumab are listed forzero-substitution homodimer (IgG1/IgG1), one-substitution pI-engineeredheterodimer (pI/IgG1), and two-substitution pI-engineered homodimer(pI/pI). The average difference in pI of the heterodimer from thehomodimers (delta pI) is also listed.

FIG. 45A-45I. Examples of basic to neutral substitutions that can bemade in the heavy chain to facilitate easy purification of aheterodimeric species. Calculated pI in the context of bevacizumab arelisted for zero-substitution homodimer (IgG1/IgG1), one-substitutionpI-engineered heterodimer (pI/IgG1), and two-substitution pI-engineeredhomodimer (pI/pI). The average difference in pI of the heterodimer fromthe homodimers (delta pI) is also listed.

FIG. 46A-46G. Examples of basic substitutions that can be made in theheavy chain to facilitate easy purification of a heterodimeric species.Calculated pI in the context of bevacizumab are listed forzero-substitution homodimer (IgG1/IgG1), one-substitution pI-engineeredheterodimer (pI/IgG1), and two-substitution pI-engineered homodimer(pI/pI). The average difference in pI of the heterodimer from thehomodimers (delta pI) is also listed.

FIG. 47A-47H. Examples of acidic to neutral substitutions that can bemade in the heavy chain to facilitate easy purification of aheterodimeric species. Calculated pI in the context of bevacizumab arelisted for zero-substitution homodimer (IgG1/IgG1), one-substitutionpI-engineered heterodimer (pI/IgG1), and two-substitution pI-engineeredhomodimer (pI/pI). The average difference in pI of the heterodimer fromthe homodimers (delta pI) is also listed.

FIG. 48A-48B. Examples of acidic substitutions that can be made in thelight chain to facilitate easy purification of a heterodimeric species.Calculated pI in the context of bevacizumab are listed forzero-substitution homodimer (IgG1/IgG1), one-substitution pI-engineeredheterodimer (pI/IgG1), and two-substitution pI-engineered homodimer(pI/pI). The average difference in pI of the heterodimer from thehomodimers (delta pI) is also listed.

FIG. 49A-49D. Examples of basic to neutral substitutions that can bemade in the light chain to facilitate easy purification of aheterodimeric species. Calculated pI in the context of bevacizumab arelisted for zero-substitution homodimer (IgG1/IgG1), one-substitutionpI-engineered heterodimer (pI/IgG1), and two-substitution pI-engineeredhomodimer (pI/pI). The average difference in pI of the heterodimer fromthe homodimers (delta pI) is also listed.

FIG. 50A-50B. Examples of basic substitutions that can be made in thelight chain to facilitate easy purification of a heterodimeric species.Calculated pI in the context of bevacizumab are listed forzero-substitution homodimer (IgG1/IgG1), one-substitution pI-engineeredheterodimer (pI/IgG1), and two-substitution pI-engineered homodimer(pI/pI). The average difference in pI of the heterodimer from thehomodimers (delta pI) is also listed.

FIG. 51A-51D. Examples of acidic to neutral substitutions that can bemade in the light chain to facilitate easy purification of aheterodimeric species. Calculated pI in the context of bevacizumab arelisted for zero-substitution homodimer (IgG1/IgG1), one-substitutionpI-engineered heterodimer (pI/IgG1), and two-substitution pI-engineeredhomodimer (pI/pI). The average difference in pI of the heterodimer fromthe homodimers (delta pI) is also listed.

FIG. 52A-52D. Sequence alignment of IgG1, IgG2, IgG3, IgG4, ISO(−),ISO(+RR), and ISO(+). For IgG1-4, differences from the IgG1 sequence arehighlighted in grey. For isotypic pI variants, differences from IgG1 areshown in black with white text.

FIG. 53A-53-B. Sequences of IOS(−), ISO(+), ISO(+RR), Anti-VEGF ISO(−),Anti-VEGF ISO(+), and Anti-VEGF ISO(+RR).

FIG. 54. Sequence of XENP10783, Anti-VEGF ISO(−)×IgG1(WT). Also listedare the three expected species and their respective pI aftertransfection and protein A purification.

FIG. 55. Sequence of XENP10784, Anti-VEGF ISO(+RR)×IgG1(WT). Also listedare the three expected species and their respective pI aftertransfection and protein A purification.

FIG. 56. Sequence of XENP10896, Anti-VEGF ISO(−)×ISO(+RR). Also listedare the three expected species and their respective pI aftertransfection and protein A purification.

FIG. 57. Sequence of XENP10901, Anti-VEGF ISO(−)×ISO(+). Also listed arethe three expected species and their respective pI after transfectionand protein A purification.

FIG. 58A-58C. List of all possible reduced pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG 1-WT heavy chain.

FIG. 59. List of all possible increased pI variants created fromisotypic substitutions of IgG1-4. Shown are the pI values for the threeexpected species as well as the average delta pI between the heterodimerand the two homodimer species present when the variant heavy chain istransfected with IgG1-WT heavy chain.

FIG. 60. Chromatogram and IEF gel demonstrating purification of theheterodimer species present when Anti-VEGF ISO(−), IgG1-WT, andAnti-VEGF WT light chain are transfected together. Purification isperformed on a HiTrap SP HP cation exchange column using 50 mM MES @ pH6.0 and eluted with a linear NaCl gradient (0-130 mM).

FIG. 61. Chromatogram and IEF gel demonstrating purification of theheterodimer species present when Anti-VEGF ISO(+RR), IgG1-WT, andAnti-VEGF WT light chain are transfected together. Purification isperformed on a HiTrap SP HP cation exchange column using 50 mM MES @ pH6.0 and eluted with a linear NaCl gradient (0-180 mM).

FIG. 62. Chromatogram and IEF gel demonstrating purification of theheterodimer species present when Anti-VEGF ISO(−), ISO(+RR), andAnti-VEGF WT light chain are transfected together. Purification isperformed on a HiTrap SP HP cation exchange column using 50 mM MES @ pH6.0 and eluted with a linear NaCl gradient (0-180 mM).

FIG. 63. Chromatogram and IEF gel demonstrating purification of theheterodimer species present when Anti-VEGF ISO(−), ISO(+), and Anti-VEGFWT light chain are transfected together. Purification is performed on aHiTrap SP HP cation exchange column using 50 mM MES @ pH 6.0 and elutedwith a linear NaCl gradient (0-180 mM).

FIG. 64. Structure and sequences of a pI-engineered variant,specifically an anti-CD 19×anti-CD3 mAb-Fv. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 65. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 scFv2-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 66. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 DART-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 67. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 scFv-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 68. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 mAb-scFv. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 69. Structure and sequences of a pI-engineered variant,specifically an anti-CD 19×anti-CD3 mAb-dAb. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 70. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 Fv-Fab-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 71. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 common light chain mAb. Thecalculated pI of heterodimeric and homodimeric species is listed.

FIG. 72. Structure and sequences of a pI-engineered variant,specifically an anti-CD3 one-arm mAb. The calculated pI of heterodimericand homodimeric species is listed.

FIG. 73. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 Fab-Fv-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 74. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 Fv-Fv-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 75. Structure and sequences of a pI-engineered variant,specifically an anti-CD3 monovalent mAb. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 76. Structure and sequences of a pI-engineered variant,specifically an anti-CD 19×anti-CD3 central mAb-Fv. The calculated pI ofheterodimeric and homodimeric species is listed.

FIG. 77. Structure and sequences of a pI-engineered variant,specifically an anti-CD19×anti-CD3 Fab-Fab-Fc. The calculated pI ofheterodimeric and homodimeric species is listed.

FIGS. 78A-78N depict a variety of heterodimerization formats. As apreliminary matter, the structures of FIG. 78 all show a fusion partnerof a variable region (including scFvs). However, as described herein forfusion proteins, other binding ligands can take the place of thesevariable regions. FIG. 78A shows a dual scFv-Fc format, that, as for allheterodimerization formats herein can include heterodimerizationvariants such as pI variants, knobs in holes (KIH, also referred toherein as steric variants), charge pairs (a subset of steric variants),and SEED body (“strand-exchange engineered domain”; see Klein et al.,mAbs 4:6 653-663 (2012) and Davis et al, Protein Eng Des Sel 201023:195-202) which rely on the fact that the CH3 domains of human IgG andIgA do not bind to each other. As for all the heterodimeric structuresherein, these heterodimerization variants can be combined, optionallyand independently and in any combination. What is important is that the“strandedness” of the monomer pairs remains intact although variantslisted as “monomer 1” variants in the steric list can be crossed with“monomer 2” variants in the pI list. That is, any set can be combinedwith any other, regardless of which “monomer” list to which they areassociated. FIG. 78B depicts a bispecific IgG, again with the option ofa variety of heterodimerization variants. FIG. 78C depicts thebispecific IgG but with the use of common light chains. FIG. 78D depictsthe “one armed” version of DVD-Ig which utilizes two different variableheavy and variable light domains. FIG. 78E is similar, except thatrather than an “empty arm”, the variable heavy and light chains are onopposite heavy chains. FIG. 78F is generally referred as “mAb-Fv”. FIG.78G depicts a multi-scFv format; as will be appreciated by those in theart, similar to the “A, B, C, D” formats depicted in FIG. 64-77, theremay be any number of associated scFvs (or, for that matter, any otherbinding ligands or functionalities). Thus, FIG. 78G could have 1, 2, 3or 4 scFvs (e.g. for bispecifics, the scFv could be “cis” or “trans”, orboth on one “end” of the molecule). FIG. 78H depicts a heterodimericFabFc with the Fab being formed by two different heavy chains onecontaining heavy chain Fab sequences and the other containing lightchain Fab sequences. FIG. 78I depicts the “one armed Fab-Fc”, where oneheavy chain comprises the Fab. FIG. 78J depicts a “one armed scFv-Fc”,wherein one heavy chain Fc comprises an scFv and the other heavy chainis “empty”. FIG. 78K shows a scFv-CH3, wherein only heavy chain CH3regions are used, each with their own scFv. FIG. 78L depicts a mAb-scFv,wherein one end of the molecule engages an antigen bivalently with amonovalent engagement using an scFv on one of the heavy chains. FIG. 78Mdepicts the same structure except that both heavy chains comprise anadditional scFv, which can either bind the same antigen or differentantigens. FIG. 78N shows the “CrossMab” structure, where the problem ofmultiplex formation due to two different light chains is addressed byswitching sequences in the Fab portion.

FIGS. 79A and 79B show novel steric variants. As will be understood bythose in the art, the first column of each table represents“corresponding” monomer pairs: that is, monomer 1 has 405A and thecorresponding steric variant is 394F.

FIG. 80A-80B depicts heterodimerization variants that find particularuse in the present invention.

FIG. 81A-81B depicts heterodimerization variants of use in the presentinvention.

FIG. 82 depicts novel pI heterodimerization variants.

FIG. 83 depicts a matrix of possible combinations of heterodimerizationformats, heterodimerization variants (separated into pI variants andsteric variants (which includes charge pair variants), Fc variants, FcRnvariants and combinations. Legend A are suitable FcRn variants: 434A,434S, 428L, 308F, 259I, 428L/434S, 259I/308F, 436I/428L, 436I or V/434S,436V/428L, 252Y, 252Y/254T/256E and 259I/308F/428L. That is, the dualscFv-Fc format of FIG. 78A can have any of these FcRn variants. Forclarity, as each heavy chain is different, FcRn variants (as well as theFc variants) can reside on one or both monomers. Legend B are suitableFc variants: 236A, 239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F,267E/328F, 236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R,236R/328R, 236N/267E, 243L, 298A and 299T. (Note, additional suitable Fcvariants are found in FIG. 41 of US 2006/0024298, the figure and legendof which are hereby incorporated by reference in their entirety). LegendC are suitable pI variants, and these, for brevity are imported fromFIG. 82, again with the understanding that there is a “strandedness” topI variants. Legend D are suitable steric variants (including chargepair variants); again, for brevity are imported from FIG. 80, again withthe understanding that there is a “strandedness” to steric variants.Legend E reflects the following possible combinations, again, with eachvariant being independently and optionally combined from the appropriatesource Legend: 1) pI variants plus FcRn variants; 2) pI variants plus Fcvariants; 3) pI variants plus FcRn variants plus Fc variants; 4) stericvariants plus FcRn variants; 5) steric variants plus Fc variants; 6)steric variants plus FcRn variants plus Fc variants; 7) pI variants plussteric variants plus FcRn variants; 8) pI variants plus steric variantsplus Fc variants; 9) pI variants plus steric variants plus FcRn variantsplus Fc variants; and 10) pI variants plus steric variants.

FIGS. 1-76 of U.S. Ser. No. 61/593,846 and the associated legends anddiscussion in the specification are hereby incorporated by reference.

FIGS. 2 and 17 of U.S. Ser. No. 61/778,157, inclusive of all thesequences including the optimized CD3 sequences are expresslyincorporated herein by reference.

DETAILED DESCRIPTION OF THE INVENTION I. Overview

The present invention is directed to novel constructs to providebispecific antibodies (or, as discussed below, trispecific ortetraspecific antibodies can also be made). An ongoing problem inantibody technologies is the desire for “bispecific” (and/ormultispecific) antibodies that bind to two (or more) different antigenssimultaneously, in general thus allowing the different antigens to bebrought into proximity and resulting in new functionalities and newtherapies. In general, these antibodies are made by including genes foreach heavy and light chain into the host cells. This generally resultsin the formation of the desired heterodimer (A-B), as well as the twohomodimers (A-A and B-B). However, a major obstacle in the formation ofmultispecific antibodies is the difficulty in purifying theheterodimeric antibodies away from the homodimeric antibodies and/orbiasing the formation of the heterodimer over the formation of thehomodimers.

The present invention is generally directed to the creation ofheterodimeric proteins including antibodies, that can co-engage antigensin several ways, relying on amino acid variants in the constant regionsthat are different on each chain to promote heterodimeric formationand/or allow for ease of purification of heterodimers over thehomodimers.

There are a number of mechanisms that can be used to generate theheterodimers of the present invention. In addition, as will beappreciated by those in the art, these mechanisms can be combined toensure high heterodimerization. Thus, amino acid variants that lead tothe production of heterodimers are referred to as “heterodimerizationvariants”. As discussed below, heterodimerization variants can includesteric variants (e.g. the “knobs and holes” variants described below andthe “charge pairs” variants described below) as well as “pI variants”,which allows purification of homodimers away from heterodimers.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation, asdescribed in U.S. Ser. No. 61/596,846 and U.S. Ser. No. 12/875,015,Ridgway et al., Protein Engineering 9(7):617 (1996); Atwell et al., J.Mol. Biol. 1997 270:26; U.S. Pat. No. 8,216,805, US 2012/0149876, all ofwhich are hereby incorporated by reference in their entirety. TheFigures identify a number of “monomer A-monomer B” pairs that rely on“knobs and holes”. In addition, as described in Merchant et al., NatureBiotech. 16:677 (1998), these “knobs and hole” mutations can be combinedwith disulfide bonds to skew formation to heterodimerization.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” or “charge pairs”as described in Gunasekaran et al., J. Biol. Chem. 285(25):19637 (2010),hereby incorporated by reference in its entirety. This is sometimesreferred to herein as “charge pairs”. In this embodiment, electrostaticsare used to skew the formation towards heterodimerization. As those inthe art will appreciate, these may also have an effect on pI, and thuson purification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

In the present invention, in some embodiments, pI variants are used toalter the pI of one or both of the monomers and thus allowing theisoelectric purification of A-A, A-B and B-B dimeric proteins.

In the present invention that utilizes pI as a separation mechanism toallow the purification of heterodimeric proteins, amino acid variantscan be introduced into one or both of the monomer polypeptides; that is,the pI of one of the monomers (referred to herein for simplicity as“monomer A”) can be engineered away from monomer B, or both monomer Aand B change be changed, with the pI of monomer A increasing and the pIof monomer B decreasing. As is outlined more fully below, the pI changesof either or both monomers can be done by removing or adding a chargedresidue (e.g. a neutral amino acid is replaced by a positively ornegatively charged amino acid residue, e.g. glycine to glutamic acid),changing a charged residue from positive or negative to the oppositecharge (aspartic acid to lysine) or changing a charged residue to aneutral residue (e.g. loss of a charge; lysine to serine.

Accordingly, in this embodiment of the present invention provides forcreating a sufficient change in pI in at least one of the monomers suchthat heterodimers can be separated from homodimers. As will beappreciated by those in the art, and as discussed further below, thiscan be done by using a “wild type” heavy chain constant region and avariant region that has been engineered to either increase or decreaseit's pI (wt A−+B or wt A−−B), or by increasing one region and decreasingthe other region (A+−B− or A−B+).

Thus, in general, a component of some embodiments of the presentinvention are amino acid variants in the constant regions of antibodiesthat are directed to altering the isoelectric point (pI) of at leastone, if not both, of the monomers of a dimeric protein to form “pIheterodimers” (when the protein is an antibody, these are referred to as“pI antibodies”) by incorporating amino acid substitutions (“pIvariants” or “pI substitutions”) into one or both of the monomers. Asshown herein, the separation of the heterodimers from the two homodimerscan be accomplished if the pIs of the two monomers differ by as littleas 0.1 pH unit, with 0.2, 0.3, 0.4 and 0.5 or greater all finding use inthe present invention.

Furthermore, as will be appreciated by those in the art and outlinedherein, in some cases, heterodimers can be separated from homodimers onthe basis of size.

By using the constant region of the heavy chain, a more modular approachto designing and purifying multispecific proteins, including antibodies,is provided. In addition, in some embodiments, the possibility ofimmunogenicity resulting from the pI variants is significantly reducedby importing pI variants from different IgG isotypes such that pI ischanged without introducing significant immunogenicity. Thus, anadditional problem to be solved is the elucidation of low pI constantdomains with high human sequence content, e.g. the minimization oravoidance of non-human residues at any particular position.

A side benefit that can occur with this pI engineering is also theextension of serum half-life and increased FcRn binding. That is, asdescribed in U.S. Ser. No. 13/194,904 (incorporated by reference in itsentirety), lowering the pI of antibody constant domains (including thosefound in antibodies and Fc fusions) can lead to longer serum retentionin vivo. These pI variants for increased serum half life also facilitatepI changes for purification.

In addition, it should be noted that the pI variants of theheterodimerization variants give an additional benefit for the analyticsand quality control process of bispecific antibodies, as, particularlyin the case of CD3 antibodies, the ability to either eliminate, minimizeand distinguish when homodimers are present is significant. Similarly,the ability to reliably test the reproducibility of the heterodimericprotein production is important.

In addition to all or part of a variant heavy constant domain, one orboth of the monomers may contain one or two fusion partners, such thatthe heterodimers form multivalent proteins. As is generally depicted theFigures, the fusion partners are depicted as A, B, C and D, with allcombinations possible. In general, A, B, C and D are selected such thatthe heterodimer is at least bispecific or bivalent in its ability tointeract with additional proteins.

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inthe FIG. 78. Some figures depict “single ended” configurations, wherethere is one type of specificity on one “arm” of the molecule and adifferent specificity on the other “arm”. Other figures depict “dualended” configurations, where there is at least one type of specificityat the “top” of the molecule and one or more different specificities atthe “bottom” of the molecule. Furthermore as is shown, these twoconfigurations can be combined, where there can be triple or quadruplespecificities based on the particular combination. Thus, the presentinvention provides “multispecific” binding proteins, includingmultispecific antibodies.

In addition, as further described below, additional amino acidsubstitutions can be engineered into the Fc region of the proteins ofthe invention, to alter a variety of additional functionalities such asaltered FcγR binding (e.g. ADCC, for example), altered FcRn binding (toalter half-life of the antibody in the serum), etc.

DEFINITIONS

In order that the application may be more completely understood, severaldefinitions are set forth below. Such definitions are meant to encompassgrammatical equivalents.

By “ablation” herein is meant a decrease or removal of activity. Thusfor example, “ablating FcγR binding” means the Fc region amino acidvariant has less than 50% starting binding as compared to an Fc regionnot containing the specific variant, with less than 70-80-90-95-98% lossof activity being preferred, and in general, with the activity beingbelow the level of detectable binding in a Biacore assay. Of particularuse in the ablation of FcγR binding is the double variant 236R/328R, and236R and 328R separately as well.

By “ADCC” or “antibody dependent cell-mediated cytotoxicity” as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause lysis of the target cell. ADCC is correlated withbinding to FcγRIIIa; increased binding to FcγRIIIa leads to an increasein ADCC activity.

By “ADCP” or antibody dependent cell-mediated phagocytosis as usedherein is meant the cell-mediated reaction wherein nonspecific cytotoxiccells that express FcγRs recognize bound antibody on a target cell andsubsequently cause phagocytosis of the target cell.

By “modification” herein is meant an amino acid substitution, insertion,and/or deletion in a polypeptide sequence or an alteration to a moietychemically linked to a protein. For example, a modification may be analtered carbohydrate or PEG structure attached to a protein. By “aminoacid modification” herein is meant an amino acid substitution,insertion, and/or deletion in a polypeptide sequence. For clarity,unless otherwise noted, the amino acid modification is always to anamino acid coded for by DNA, e.g. the 20 amino acids that have codons inDNA and RNA.

By “amino acid substitution” or “substitution” herein is meant thereplacement of an amino acid at a particular position in a parentpolypeptide sequence with a different amino acid. In particular, in someembodiments, the substitution is to an amino acid that is not naturallyoccurring at the particular position, either not naturally occurringwithin the organism or in any organism. For example, the substitutionE272Y refers to a variant polypeptide, in this case an Fc variant, inwhich the glutamic acid at position 272 is replaced with tyrosine. Forclarity, a protein which has been engineered to change the nucleic acidcoding sequence but not change the starting amino acid (for exampleexchanging CGG (encoding arginine) to CGA (still encoding arginine) toincrease host organism expression levels) is not an “amino acidsubstitution”; that is, despite the creation of a new gene encoding thesame protein, if the protein has the same amino acid at the particularposition that it started with, it is not an amino acid substitution.

By “amino acid insertion” or “insertion” as used herein is meant theaddition of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, −233E or 233E designates an insertionof glutamic acid after position 233 and before position 234.Additionally, −233ADE or A233ADE designates an insertion of AlaAspGluafter position 233 and before position 234.

By “amino acid deletion” or “deletion” as used herein is meant theremoval of an amino acid sequence at a particular position in a parentpolypeptide sequence. For example, E233− or E233# designates a deletionof glutamic acid at position 233. Additionally, EDA233− or EDA233#designates a deletion of the sequence GluAspAla that begins at position233.

By “variant protein” or “protein variant”, or “variant” as used hereinis meant a protein that differs from that of a parent protein by virtueof at least one amino acid modification. Protein variant may refer tothe protein itself, a composition comprising the protein, or the aminosequence that encodes it. Preferably, the protein variant has at leastone amino acid modification compared to the parent protein, e.g. fromabout one to about seventy amino acid modifications, and preferably fromabout one to about five amino acid modifications compared to the parent.As described below, in some embodiments the parent polypeptide, forexample an Fc parent polypeptide, is a human wild type sequence, such asthe Fc region from IgG1, IgG2, IgG3 or IgG4, although human sequenceswith variants can also serve as “parent polypeptides”. The proteinvariant sequence herein will preferably possess at least about 80%identity with a parent protein sequence, and most preferably at leastabout 90% identity, more preferably at least about 95-98-99% identity.Variant protein can refer to the variant protein itself, compositionscomprising the protein variant, or the DNA sequence that encodes it.Accordingly, by “antibody variant” or “variant antibody” as used hereinis meant an antibody that differs from a parent antibody by virtue of atleast one amino acid modification, “IgG variant” or “variant IgG” asused herein is meant an antibody that differs from a parent IgG (again,in many cases, from a human IgG sequence) by virtue of at least oneamino acid modification, and “immunoglobulin variant” or “variantimmunoglobulin” as used herein is meant an immunoglobulin sequence thatdiffers from that of a parent immunoglobulin sequence by virtue of atleast one amino acid modification. “Fc variant” or “variant Fc” as usedherein is meant a protein comprising an amino acid modification in an Fcdomain. The Fc variants of the present invention are defined accordingto the amino acid modifications that compose them. Thus, for example,N434S or 434S is an Fc variant with the substitution serine at position434 relative to the parent Fc polypeptide, wherein the numbering isaccording to the EU index. Likewise, M428L/N434S defines an Fc variantwith the substitutions M428L and N434S relative to the parent Fcpolypeptide. The identity of the WT amino acid may be unspecified, inwhich case the aforementioned variant is referred to as 428L/4345. It isnoted that the order in which substitutions are provided is arbitrary,that is to say that, for example, 428L/4345 is the same Fc variant asM428L/N434S, and so on. For all positions discussed in the presentinvention that relate to antibodies, unless otherwise noted, amino acidposition numbering is according to the EU index. The EU index or EUindex as in Kabat or EU numbering scheme refers to the numbering of theEU antibody (Edelman et al., 1969, Proc Natl Acad Sci USA 63:78-85,hereby entirely incorporated by reference.) The modification can be anaddition, deletion, or substitution. Substitutions can include naturallyoccurring amino acids and, in some cases, synthetic amino acids.Examples include U.S. Pat. No. 6,586,207; WO 98/48032; WO 03/073238;US2004-0214988A1; WO 05/35727A2; WO 05/74524A2; J. W. Chin et al.,(2002), Journal of the American Chemical Society 124:9026-9027; J. W.Chin, & P. G. Schultz, (2002), ChemBioChem 11:1135-1137; J. W. Chin, etal., (2002), PICAS United States of America 99:11020-11024; and, L.Wang, & P. G. Schultz, (2002), Chem. 1-10, all entirely incorporated byreference.

As used herein, “protein” herein is meant at least two covalentlyattached amino acids, which includes proteins, polypeptides,oligopeptides and peptides. The peptidyl group may comprise naturallyoccurring amino acids and peptide bonds, or synthetic peptidomimeticstructures, i.e. “analogs”, such as peptoids (see Simon et al., PNAS USA89(20):9367 (1992), entirely incorporated by reference). The amino acidsmay either be naturally occurring or synthetic (e.g. not an amino acidthat is coded for by DNA); as will be appreciated by those in the art.For example, homo-phenylalanine, citrulline, ornithine and norleucineare considered synthetic amino acids for the purposes of the invention,and both D- and L-(R or S) configured amino acids may be utilized. Thevariants of the present invention may comprise modifications thatinclude the use of synthetic amino acids incorporated using, forexample, the technologies developed by Schultz and colleagues, includingbut not limited to methods described by Cropp & Shultz, 2004, TrendsGenet. 20(12):625-30, Anderson et al., 2004, Proc Natl Acad Sci USA 101(2):7566-71, Zhang et al., 2003, 303(5656):371-3, and Chin et al., 2003,Science 301(5635):964-7, all entirely incorporated by reference. Inaddition, polypeptides may include synthetic derivatization of one ormore side chains or termini, glycosylation, PEGylation, circularpermutation, cyclization, linkers to other molecules, fusion to proteinsor protein domains, and addition of peptide tags or labels.

By “residue” as used herein is meant a position in a protein and itsassociated amino acid identity. For example, Asparagine 297 (alsoreferred to as Asn297 or N297) is a residue at position 297 in the humanantibody IgG1.

By “Fab” or “Fab region” as used herein is meant the polypeptide thatcomprises the VH, CHL VL, and CL immunoglobulin domains. Fab may referto this region in isolation, or this region in the context of a fulllength antibody, antibody fragment or Fab fusion protein. By “Fv” or “Fvfragment” or “Fv region” as used herein is meant a polypeptide thatcomprises the VL and VH domains of a single antibody.

By “IgG subclass modification” or “isotype modification” as used hereinis meant an amino acid modification that converts one amino acid of oneIgG isotype to the corresponding amino acid in a different, aligned IgGisotype. For example, because IgG1 comprises a tyrosine and IgG2 aphenylalanine at EU position 296, a F296Y substitution in IgG2 isconsidered an IgG subclass modification.

By “non-naturally occurring modification” as used herein is meant anamino acid modification that is not isotypic. For example, because noneof the IgGs comprise a serine at position 434, the substitution 434S inIgG1, IgG2, IgG3, or IgG4 (or hybrids thereof) is considered anon-naturally occurring modification.

By “amino acid” and “amino acid identity” as used herein is meant one ofthe 20 naturally occurring amino acids that are coded for by DNA andRNA.

By “effector function” as used herein is meant a biochemical event thatresults from the interaction of an antibody Fc region with an Fcreceptor or ligand. Effector functions include but are not limited toADCC, ADCP, and CDC.

By “IgG Fc ligand” as used herein is meant a molecule, preferably apolypeptide, from any organism that binds to the Fc region of an IgGantibody to form an Fc/Fc ligand complex. Fc ligands include but are notlimited to FcγRIs, FcγRIIs, FcγRIIIs, FcRn, C1q, C3, mannan bindinglectin, mannose receptor, staphylococcal protein A, streptococcalprotein G, and viral FcγR. Fc ligands also include Fc receptor homologs(FcRH), which are a family of Fc receptors that are homologous to theFcγRs (Davis et al., 2002, Immunological Reviews 190:123-136, entirelyincorporated by reference). Fc ligands may include undiscoveredmolecules that bind Fc. Particular IgG Fc ligands are FcRn and Fc gammareceptors. By “Fc ligand” as used herein is meant a molecule, preferablya polypeptide, from any organism that binds to the Fc region of anantibody to form an Fc/Fc ligand complex.

By “Fc gamma receptor”, “FcγR” or “FcqammaR” as used herein is meant anymember of the family of proteins that bind the IgG antibody Fc regionand is encoded by an FcγR gene. In humans this family includes but isnot limited to FcγRI (CD64), including isoforms FcγRIa, FcγRIb, andFcγRIc; FcγRII (CD32), including isoforms FcγRIIa (including allotypesH131 and R131), FcγRIIb (including FcγRIIb-1 and FcγRIIb-2), andFcγRIIc; and FcγRIII (CD16), including isoforms FcγRIIIa (includingallotypes V158 and F158) and FcγRIIIb (including allotypes FcγRIIb-NA1and FcγRIIb-NA2) (Jefferis et al., 2002, Immunol Lett 82:57-65, entirelyincorporated by reference), as well as any undiscovered human FcγRs orFcγR isoforms or allotypes. An FcγR may be from any organism, includingbut not limited to humans, mice, rats, rabbits, and monkeys. Mouse FcγRsinclude but are not limited to FcγRI (CD64), FcγRII (CD32), FcγRIII(CD16), and FcγRIII-2 (CD16-2), as well as any undiscovered mouse FcγRsor FcγR isoforms or allotypes.

By “FcRn” or “neonatal Fc Receptor” as used herein is meant a proteinthat binds the IgG antibody Fc region and is encoded at least in part byan FcRn gene. The FcRn may be from any organism, including but notlimited to humans, mice, rats, rabbits, and monkeys. As is known in theart, the functional FcRn protein comprises two polypeptides, oftenreferred to as the heavy chain and light chain. The light chain isbeta-2-microglobulin and the heavy chain is encoded by the FcRn gene.Unless otherwise noted herein, FcRn or an FcRn protein refers to thecomplex of FcRn heavy chain with beta-2-microglobulin. A variety of FcRnvariants used to increase binding to the FcRn receptor, and in somecases, to increase serum half-life, are shown in the Figure Legend ofFIG. 83.

By “parent polypeptide” as used herein is meant a starting polypeptidethat is subsequently modified to generate a variant. The parentpolypeptide may be a naturally occurring polypeptide, or a variant orengineered version of a naturally occurring polypeptide. Parentpolypeptide may refer to the polypeptide itself, compositions thatcomprise the parent polypeptide, or the amino acid sequence that encodesit. Accordingly, by “parent immunoglobulin” as used herein is meant anunmodified immunoglobulin polypeptide that is modified to generate avariant, and by “parent antibody” as used herein is meant an unmodifiedantibody that is modified to generate a variant antibody. It should benoted that “parent antibody” includes known commercial, recombinantlyproduced antibodies as outlined below.

By “Fc fusion protein” or “immunoadhesin” herein is meant a proteincomprising an Fc region, generally linked (optionally through a linkermoiety, as described herein) to a different protein, such as a bindingmoiety to a target protein, as described herein).

By “position” as used herein is meant a location in the sequence of aprotein. Positions may be numbered sequentially, or according to anestablished format, for example the EU index for antibody numbering.

By “target antigen” as used herein is meant the molecule that is boundspecifically by the variable region of a given antibody. A targetantigen may be a protein, carbohydrate, lipid, or other chemicalcompound. A wide number of suitable target antigens are described below.

By “strandedness” in the context of the monomers of the heterodimericproteins of the invention herein is meant that, similar to the twostrands of DNA that “match”, heterodimerization variants areincorporated into each monomer so as to preserve the ability to “match”to form heterodimers. For example, if some pI variants are engineeredinto monomer A (e.g. making the pI higher) then steric variants that are“charge pairs” that can be utilized as well do not interfere with the pIvariants, e.g. the charge variants that make a pI higher are put on thesame “strand” or “monomer” to preserve both functionalities.

By “target cell” as used herein is meant a cell that expresses a targetantigen.

By “variable region” as used herein is meant the region of animmunoglobulin that comprises one or more Ig domains substantiallyencoded by any of the V.kappa., V.lamda., and/or VH genes that make upthe kappa, lambda, and heavy chain immunoglobulin genetic locirespectively.

By “wild type or WT” herein is meant an amino acid sequence or anucleotide sequence that is found in nature, including allelicvariations. A WT protein has an amino acid sequence or a nucleotidesequence that has not been intentionally modified.

Heterodimeric Proteins

The present invention is directed to the generation of multispecific,particularly bispecific binding proteins, and in particular,multispecific antibodies.

Antibodies

The present invention relates to the generation of heterodimericantibodies, generally therapeutic antibodies, through the use of“heterodimerization amino acid variants”. As is discussed below, theterm “antibody” is used generally. Antibodies that find use in thepresent invention can take on a number of formats as described herein,including traditional antibodies as well as antibody derivatives,fragments and mimetics, described below. In general, the term “antibody”includes any polypeptide that includes at least one constant domain,including, but not limited to, CHL CH2, CH3 and CL.

Traditional antibody structural units typically comprise a tetramer.Each tetramer is typically composed of two identical pairs ofpolypeptide chains, each pair having one “light” (typically having amolecular weight of about 25 kDa) and one “heavy” chain (typicallyhaving a molecular weight of about 50-70 kDa). Human light chains areclassified as kappa and lambda light chains. The present invention isdirected to the IgG class, which has several subclasses, including, butnot limited to IgG1, IgG2, IgG3, and IgG4. Thus, “isotype” as usedherein is meant any of the subclasses of immunoglobulins defined by thechemical and antigenic characteristics of their constant regions. Itshould be understood that therapeutic antibodies can also comprisehybrids of isotypes and/or subclasses. For example, as shown herein, thepresent invention covers heterodimers that can contain one or bothchains that are IgG1/G2 hybrids (see SEQ ID NO:6, for example).

The amino-terminal portion of each chain includes a variable region ofabout 100 to 110 or more amino acids primarily responsible for antigenrecognition, generally referred to in the art and herein as the “Fvdomain” or “Fv region”. In the variable region, three loops are gatheredfor each of the V domains of the heavy chain and light chain to form anantigen-binding site. Each of the loops is referred to as acomplementarity-determining region (hereinafter referred to as a “CDR”),in which the variation in the amino acid sequence is most significant.“Variable” refers to the fact that certain segments of the variableregion differ extensively in sequence among antibodies. Variabilitywithin the variable region is not evenly distributed. Instead, the Vregions consist of relatively invariant stretches called frameworkregions (FRs) of 15-30 amino acids separated by shorter regions ofextreme variability called “hypervariable regions” that are each 9-15amino acids long or longer.

Each VH and VL is composed of three hypervariable regions(“complementary determining regions,” “CDRs”) and four FRs, arrangedfrom amino-terminus to carboxy-terminus in the following order:FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4.

The hypervariable region generally encompasses amino acid residues fromabout amino acid residues 24-34 (LCDR1; “L” denotes light chain), 50-56(LCDR2) and 89-97 (LCDR3) in the light chain variable region and aroundabout 31-35B (HCDR1; “H” denotes heavy chain), 50-65 (HCDR2), and 95-102(HCDR3) in the heavy chain variable region; Kabat et al., SEQUENCES OFPROTEINS OF IMMUNOLOGICAL INTEREST, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991) and/or thoseresidues forming a hypervariable loop (e.g. residues 26-32 (LCDR1),50-52 (LCDR2) and 91-96 (LCDR3) in the light chain variable region and26-32 (HCDR1), 53-55 (HCDR2) and 96-101 (HCDR3) in the heavy chainvariable region; Chothia and Lesk (1987) J. Mol. Biol. 196:901-917.Specific CDRs of the invention are described below.

Throughout the present specification, the Kabat numbering system isgenerally used when referring to a residue in the variable domain(approximately, residues 1-107 of the light chain variable region andresidues 1-113 of the heavy chain variable region) (e.g, Kabat et al.,supra (1991)).

The CDRs contribute to the formation of the antigen-binding, or morespecifically, epitope binding site of antibodies. “Epitope” refers to adeterminant that interacts with a specific antigen binding site in thevariable region of an antibody molecule known as a paratope. Epitopesare groupings of molecules such as amino acids or sugar side chains andusually have specific structural characteristics, as well as specificcharge characteristics. A single antigen may have more than one epitope.

The epitope may comprise amino acid residues directly involved in thebinding (also called immunodominant component of the epitope) and otheramino acid residues, which are not directly involved in the binding,such as amino acid residues which are effectively blocked by thespecifically antigen binding peptide; in other words, the amino acidresidue is within the footprint of the specifically antigen bindingpeptide.

Epitopes may be either conformational or linear. A conformationalepitope is produced by spatially juxtaposed amino acids from differentsegments of the linear polypeptide chain. A linear epitope is oneproduced by adjacent amino acid residues in a polypeptide chain.Conformational and nonconformational epitopes may be distinguished inthat the binding to the former but not the latter is lost in thepresence of denaturing solvents.

An epitope typically includes at least 3, and more usually, at least 5or 8-10 amino acids in a unique spatial conformation. Antibodies thatrecognize the same epitope can be verified in a simple immunoassayshowing the ability of one antibody to block the binding of anotherantibody to a target antigen, for example “binning.”

In some embodiments, the antibodies are full length. By “full lengthantibody” herein is meant the structure that constitutes the naturalbiological form of an antibody, including variable and constant regions,including one or more modifications as outlined herein.

Alternatively, the antibodies can be a variety of structures, including,but not limited to, antibody fragments, monoclonal antibodies,bispecific antibodies, minibodies, domain antibodies, syntheticantibodies (sometimes referred to herein as “antibody mimetics”),chimeric antibodies, humanized antibodies, antibody fusions (sometimesreferred to as “antibody conjugates”), and fragments of each,respectively.

Antibody Fragments

In one embodiment, the antibody is an antibody fragment. Of particularinterest are antibodies that comprise Fc regions, Fc fusions, and theconstant region of the heavy chain (CH1-hinge-CH2-CH3), again alsoincluding constant heavy region fusions.

Specific antibody fragments include, but are not limited to, (i) the Fabfragment consisting of VL, VH, CL and CH1 domains, (ii) the Fd fragmentconsisting of the VH and CH1 domains, (iii) the Fv fragment consistingof the VL and VH domains of a single antibody; (iv) the dAb fragment(Ward et al., 1989, Nature 341:544-546, entirely incorporated byreference) which consists of a single variable, (v) isolated CDRregions, (vi) F(ab′)2 fragments, a bivalent fragment comprising twolinked Fab fragments (vii) single chain Fv molecules (scFv), wherein aVH domain and a VL domain are linked by a peptide linker which allowsthe two domains to associate to form an antigen binding site (Bird etal., 1988, Science 242:423-426, Huston et al., 1988, Proc. Natl. Acad.Sci. U.S.A. 85:5879-5883, entirely incorporated by reference), (viii)bispecific single chain Fv (WO 03/11161, hereby incorporated byreference) and (ix) “diabodies” or “triabodies”, multivalent ormultispecific fragments constructed by gene fusion (Tomlinson et. al.,2000, Methods Enzymol. 326:461-479; WO94/13804; Holliger et al., 1993,Proc. Natl. Acad. Sci. U.S.A. 90:6444-6448, all entirely incorporated byreference). The antibody fragments may be modified. For example, themolecules may be stabilized by the incorporation of disulphide bridgeslinking the VH and VL domains (Reiter et al., 1996, Nature Biotech.14:1239-1245, entirely incorporated by reference).

Chimeric and Humanized Antibodies

In some embodiments, the scaffold components can be a mixture fromdifferent species. As such, if the protein is an antibody, such antibodymay be a chimeric antibody and/or a humanized antibody. In general, both“chimeric antibodies” and “humanized antibodies” refer to antibodiesthat combine regions from more than one species. For example, “chimericantibodies” traditionally comprise variable region(s) from a mouse (orrat, in some cases) and the constant region(s) from a human. “Humanizedantibodies” generally refer to non-human antibodies that have had thevariable-domain framework regions swapped for sequences found in humanantibodies. Generally, in a humanized antibody, the entire antibody,except the CDRs, is encoded by a polynucleotide of human origin or isidentical to such an antibody except within its CDRs. The CDRs, some orall of which are encoded by nucleic acids originating in a non-humanorganism, are grafted into the beta-sheet framework of a human antibodyvariable region to create an antibody, the specificity of which isdetermined by the engrafted CDRs. The creation of such antibodies isdescribed in, e.g., WO 92/11018, Jones, 1986, Nature 321:522-525,Verhoeyen et al., 1988, Science 239:1534-1536, all entirely incorporatedby reference. “Backmutation” of selected acceptor framework residues tothe corresponding donor residues is often required to regain affinitythat is lost in the initial grafted construct (U.S. Pat. No. 5,530,101;U.S. Pat. No. 5,585,089; U.S. Pat. No. 5,693,761; U.S. Pat. No.5,693,762; U.S. Pat. No. 6,180,370; U.S. Pat. No. 5,859,205; U.S. Pat.No. 5,821,337; U.S. Pat. No. 6,054,297; U.S. Pat. No. 6,407,213, allentirely incorporated by reference). The humanized antibody optimallyalso will comprise at least a portion of an immunoglobulin constantregion, typically that of a human immunoglobulin, and thus willtypically comprise a human Fc region. Humanized antibodies can also begenerated using mice with a genetically engineered immune system. Roqueet al., 2004, Biotechnol. Prog. 20:639-654, entirely incorporated byreference. A variety of techniques and methods for humanizing andreshaping non-human antibodies are well known in the art (See Tsurushita& Vasquez, 2004, Humanization of Monoclonal Antibodies, MolecularBiology of B Cells, 533-545, Elsevier Science (USA), and referencescited therein, all entirely incorporated by reference). Humanizationmethods include but are not limited to methods described in Jones etal., 1986, Nature 321:522-525; Riechmann et al., 1988; Nature332:323-329; Verhoeyen et al., 1988, Science, 239:1534-1536; Queen etal., 1989, Proc Natl Acad Sci, USA 86:10029-33; He et al., 1998, J.Immunol. 160: 1029-1035; Carter et al., 1992, Proc Natl Acad Sci USA89:4285-9, Presta et al., 1997, Cancer Res. 57(20):4593-9; Gorman etal., 1991, Proc. Natl. Acad. Sci. USA 88:4181-4185; O'Connor et al.,1998, Protein Eng 11:321-8, all entirely incorporated by reference.Humanization or other methods of reducing the immunogenicity of nonhumanantibody variable regions may include resurfacing methods, as describedfor example in Roguska et al., 1994, Proc. Natl. Acad. Sci. USA91:969-973, entirely incorporated by reference. In one embodiment, theparent antibody has been affinity matured, as is known in the art.Structure-based methods may be employed for humanization and affinitymaturation, for example as described in U.S. Ser. No. 11/004,590.Selection based methods may be employed to humanize and/or affinitymature antibody variable regions, including but not limited to methodsdescribed in Wu et al., 1999, J. Mol. Biol. 294:151-162; Baca et al.,1997, J. Biol. Chem. 272(16):10678-10684; Rosok et al., 1996, J. Biol.Chem. 271(37): 22611-22618; Rader et al., 1998, Proc. Natl. Acad. Sci.USA 95: 8910-8915; Krauss et al., 2003, Protein Engineering16(10):753-759, all entirely incorporated by reference. Otherhumanization methods may involve the grafting of only parts of the CDRs,including but not limited to methods described in U.S. Ser. No.09/810,510; Tan et al., 2002, J. Immunol. 169:1119-1125; De Pascalis etal., 2002, J. Immunol. 169:3076-3084, all entirely incorporated byreference.

In one embodiment, the antibody is a minibody. Minibodies are minimizedantibody-like proteins comprising a scFv joined to a CH3 domain. Hu etal., 1996, Cancer Res. 56:3055-3061, entirely incorporated by reference.In some cases, the scFv can be joined to the Fc region, and may includesome or the entire hinge region.

Fc Fusion Heterodimeric Proteins

In addition to heterodimeric antibody constructs, the invention furtherprovides Fc fusion heterodimeric proteins. That is, rather than have theFc domain of an antibody joined to an antibody variable region, the Fcdomain can be joined to other moieties, particularly binding moietiessuch as ligands. By “Fc fusion” as used herein is meant a proteinwherein one or more polypeptides is operably linked to an Fc region. Fcfusion is herein meant to be synonymous with the terms “immunoadhesin”,“Ig fusion”, “Ig chimera”, and “receptor globulin” (sometimes withdashes) as used in the prior art (Chamow et al., 1996, Trends Biotechnol14:52-60; Ashkenazi et al., 1997, Curr Opin Immunol 9:195-200, bothentirely incorporated by reference). An Fc fusion combines the Fc regionof an immunoglobulin with a fusion partner, which in general can be anyprotein or small molecule. Virtually any protein or small molecule maybe linked to Fc to generate an Fc fusion. Protein fusion partners mayinclude, but are not limited to, the variable region of any antibody,the target-binding region of a receptor, an adhesion molecule, a ligand,an enzyme, a cytokine, a chemokine, or some other protein or proteindomain. Small molecule fusion partners may include any therapeutic agentthat directs the Fc fusion to a therapeutic target. Such targets may beany molecule, preferably an extracellular receptor, which is implicatedin disease. Thus, the IgG variants can be linked to one or more fusionpartners.

Thus, while many embodiments herein depict antibody components such asvariable heavy and light chains or scFvs, other binding moeities can befused to Fc regions to form heterodimeric proteins. For example, asdiscussed in Kontermann, supra, any number of dual targeting strategiescan be done. For example (assuming only two binding moieties perheterodimer, e.g generally one per monomer), both monomers can bindand/or neutralize two ligands or two receptors, or bind and activate twoligands or two receptors. Similarly, one monomer may bind a receptor andthe other a ligand (again, independently activating or neutralizing thebinding partner). Further, each monomer may bind to same receptor orligand in different locations (e.g. different epitopes). See FIG. 1 ofKontermann, expressly incorporated by reference. Suitable receptors andligands are outlined below in the “Target” section.

Heterodimerization Variants

Accordingly, the present invention provides heterodimeric proteins basedon the use of monomers containing variant heavy chain constant regionsas a first domain. By “monomer” herein is meant one half of theheterodimeric protein. It should be noted that antibodies are actuallytetrameric (two heavy chains and two light chains). In the context ofthe present invention, as applicable, one pair of heavy-light chains isconsidered a “monomer”. In the case where an Fv region is one fusionpartner (e.g. heavy and light chain) and a non-antibody protein isanother fusion partner, each “half” is considered a monomer.Essentially, each monomer comprises sufficient heavy chain constantregion to allow heterodimerization engineering, whether that be all theconstant region, e.g. Ch1-hinge-CH2-CH3, the Fc region (CH2-CH3).

The variant heavy chain constant regions can comprise all or part of theheavy chain constant region, including the full length construct,CH1-hinge-CH2-CH3, or portions thereof, including for example CH2-CH3.In addition, the heavy chain region of each monomer can be the samebackbone (CH1-hinge-CH2-CH3 or CH2-CH3) or different. N- and C-terminaltruncations and additions are also included within the definition; forexample, some pI variants include the addition of charged amino acids tothe C-terminus of the heavy chain domain.

Furthermore, in addition to the pI substitutions outlined herein, theheavy chain regions may also contain additional amino acidsubstitutions, including changes for altering Fc binding as discussedbelow.

In addition, some monomers can utilize linkers between the variant heavychain constant region and the fusion partner. Traditional peptidelinkers can be used, including flexible linkers of glycine and serine.In some cases, the linkers for use as components of the monomer aredifferent from those defined below for the ADC constructs, and are inmany embodiments not cleavable linkers (such as those susceptible toproteases), although cleavable linkers may find use in some embodiments.

The heterodimerization variants include a number of different types ofvariants, including, but not limited to, steric variants, pI variants,and other variants (e.g. charge variants), that can be optionally andindependently combined with any other variants. In these embodiments, itis important to match “monomer A” with “monomer B”; that is, if aheterodimeric protein relies on both steric variants and pI variants,these need to be correctly matched to each monomer: e.g. the set ofsteric variants that work (1 set on monomer A, 1 set on monomer B) iscombined with pI variant sets (1 set on monomer A, 1 set on monomer B),such that the variants on each monomer are designed to achieve thedesired function.

Steric Variants

In some embodiments, the formation of heterodimers can be facilitated bythe addition of steric variants. That is, by changing amino acids ineach heavy chain, different heavy chains are more likely to associate toform the heterodimeric structure than to form homodimers with the sameFc amino acid sequences. Suitable steric variants are shown in theFigures, particularly FIGS. 79, 80 and 81.

One mechanism is generally referred to in the art as “knobs and holes”,referring to amino acid engineering that creates steric influences tofavor heterodimeric formation and disfavor homodimeric formation canalso optionally be used; this is sometimes referred to as “knobs andholes”, as described in U.S. Ser. No. 61/596,846, Ridgway et al.,Protein Engineering 9(7):617 (1996); Atwell et al., J. Mol. Biol. 1997270:26; U.S. Pat. No. 8,216,805, all of which are hereby incorporated byreference in their entirety. FIG. 4, further described below, identifiesa number of “monomer A-monomer B” pairs that rely on “knobs and holes”.In addition, as described in Merchant et al., Nature Biotech. 16:677(1998), these “knobs and hole” mutations can be combined with disulfidebonds to skew formation to heterodimerization. Some of these variantsare shown in FIGS. 79A, 79B, 80 and 81.

An additional mechanism that finds use in the generation of heterodimersis sometimes referred to as “electrostatic steering” as described inGunasekaran et al., J. Biol. Chem. 285(25):19637 (2010), herebyincorporated by reference in its entirety. This is sometimes referred toherein as “charge pairs”. In this embodiment, electrostatics are used toskew the formation towards heterodimerization. As those in the art willappreciate, these may also have an effect on pI, and thus onpurification, and thus could in some cases also be considered pIvariants. However, as these were generated to force heterodimerizationand were not used as purification tools, they are classified as “stericvariants”. These include, but are not limited to, D221E/P228E/L368Epaired with D221R/P228R/K409R (e.g. these are “monomer correspondingsets) and C220E/P228E/368E paired with C220R/E224R/P228R/K409R.

Additional monomer A and monomer B variants that can be combined withother variants, optionally and independently in any amount, such as pIvariants outlined herein or other steric variants that are shown in FIG.37 of US 2012/0149876, the figure and legend of which are incorporatedexpressly by reference herein.

In some embodiments, the steric variants outlined herein can beoptionally and independently incorporated with any pI variant (or othervariants such as Fc variants, FcRn variants, etc.) into one or bothmonomers.

pI Variants for Heterodimers

In general, as will be appreciated by those in the art, there are twogeneral categories of pI variants: those that increase the pI of theprotein (basic changes) and those that decrease the pI of the protein(acidic changes). As described herein, all combinations of thesevariants can be done: one monomer may be wild type, or a variant thatdoes not display a significantly different pI from wild-type, and theother can be either more basic or more acidic. Alternatively, eachmonomer is changed, one to more basic and one to more acidic.

Preferred combinations of pI variants are shown in FIG. 82.

Heavy Chain Acidic pI Changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more positive (e.g. lower the pI), one or more ofthe following substitutions can be made: S119E, K133E, K133Q, T164E,K205E, K205Q, N208D, K210E, K210Q, K274E, K320E, K322E, K326E, K334E,R355E, K392E, a deletion of K447, adding peptide DEDE at the c-terminus,G137E, N203D, K274Q, R355Q, K392N and Q419E. As outlined herein andshown in the figures, these changes are shown relative to IgG1, but allisotypes can be altered this way, as well as isotype hybrids.

In the case where the heavy chain constant domain is from IgG2-4, R133Eand R133Q can also be used.

Basic pI Changes

Accordingly, when one monomer comprising a variant heavy chain constantdomain is to be made more negative (e.g. increase the pI), one or moreof the following substitutions can be made: Q196K, P217R, P228R, N276Kand H435R. As outlined herein and shown in the figures, these changesare shown relative to IgG1, but all isotypes can be altered this way, aswell as isotype hybrids.

Antibody Heterodimers Light Chain Variants

In the case of antibody based heterodimers, e.g. where at least one ofthe monomers comprises a light chain in addition to the heavy chaindomain, pI variants can also be made in the light chain. Amino acidsubstitutions for lowering the pI of the light chain include, but arenot limited to, K126E, K126Q, K145E, K145Q, N152D, S156E, K169E, S202E,K207E and adding peptide DEDE at the c-terminus of the light chain.Changes in this category based on the constant lambda light chaininclude one or more substitutions at R108Q, Q124E, K126Q, N138D, K145Tand Q199E. In addition, increasing the pI of the light chains can alsobe done.

Isotypic Variants

In addition, many embodiments of the invention rely on the “importation”of pI amino acids at particular positions from one IgG isotype intoanother, thus reducing or eliminating the possibility of unwantedimmunogenicity being introduced into the variants. That is, IgG1 is acommon isotype for therapeutic antibodies for a variety of reasons,including high effector function. However, the heavy constant region ofIgG 1 has a higher pI than that of IgG2 (8.10 versus 7.31). Byintroducing IgG2 residues at particular positions into the IgG1backbone, the pI of the resulting monomer is lowered (or increased) andadditionally exhibits longer serum half-life. For example, IgG1 has aglycine (pI 5.97) at position 137, and IgG2 has a glutamic acid (pI3.22); importing the glutamic acid will affect the pI of the resultingprotein. As is described below, a number of amino acid substitutions aregenerally required to significant affect the pI of the variant antibody.However, it should be noted as discussed below that even changes in IgG2molecules allow for increased serum half-life.

In other embodiments, non-isotypic amino acid changes are made, eitherto reduce the overall charge state of the resulting protein (e.g. bychanging a higher pI amino acid to a lower pI amino acid), or to allowaccommodations in structure for stability, etc. as is more furtherdescribed below.

In addition, by pI engineering both the heavy and light constantdomains, significant changes in each monomer of the heterodimer can beseen. As discussed herein, having the pIs of the two monomers differ byat least 0.5 can allow separation.

Calculating pI

The pI of each monomer can depend on the pI of the variant heavy chainconstant domain and the pI of the total monomer, including the variantheavy chain constant domain and the fusion partner. Thus, in someembodiments, the change in pI is calculated on the basis of the variantheavy chain constant domain, using the chart in the Figures.Alternatively, the pI of each monomer can be compared.

pI Variants that Also Confer Better FcRn In Vitro Binding

In the case where the pI variant decreases the pI of the monomer, theycan have the added benefit of improving serum retention in vivo.

Although still under examination, Fc regions are believed to have longerhalf-lives in vivo, because binding to FcRn at pH 6 in an endosomesequesters the Fc (Ghetie and Ward, 1997 Immunol Today. 18(12): 592-598,entirely incorporated by reference). The endosomal compartment thenrecycles the Fc to the cell surface. Once the compartment opens to theextracellular space, the higher pH, ˜7.4, induces the release of Fc backinto the blood. In mice, Dall' Acqua et al. showed that Fc mutants withincreased FcRn binding at pH 6 and pH 7.4 actually had reduced serumconcentrations and the same half life as wild-type Fc (Dall' Acqua etal. 2002, J. Immunol. 169:5171-5180, entirely incorporated byreference). The increased affinity of Fc for FcRn at pH 7.4 is thoughtto forbid the release of the Fc back into the blood. Therefore, the Fcmutations that will increase Fc's half-life in vivo will ideallyincrease FcRn binding at the lower pH while still allowing release of Fcat higher pH. The amino acid histidine changes its charge state in thepH range of 6.0 to 7.4. Therefore, it is not surprising to find Hisresidues at important positions in the Fc/FcRn complex.

Recently it has been suggested that antibodies with variable regionsthat have lower isoelectric points may also have longer serum half-lives(Igawa et al., 2010 PEDS. 23(5): 385-392, entirely incorporated byreference). However, the mechanism of this is still poorly understood.Moreover, variable regions differ from antibody to antibody. Constantregion variants with reduced pI and extended half-life would provide amore modular approach to improving the pharmacokinetic properties ofantibodies, as described herein.

Combination of Heterodimeric Variants

As will be appreciated by those in the art, all of the recitedheterodimerization variants can be optionally and independently combinedin any way, as long as they retain their “strandedness” or “monomerpartition”. In addition, all of these variants can be combined into anyof the hterodimerization formats. See FIG. 83.

In the case of pI variants, while embodiments finding particular use areshown in the Figures, other combinations can be generated, following thebasic rule of altering the pI difference between two monomers tofacilitate purification.

Suitable Multispecific Formats

As will be appreciated by those in the art, there are a wide variety ofpossible multispecific formats that find use in the present invention,see for example Kontermann, mAbs 4(2):182-197 (2012), herebyincorporated by reference in its entirety and particularly Tables 1 and2 and FIGS. 1 and 2, with specific reference to the constructs ofKontermann that contain an Fc region. See also Klein et al., Of use inthe present invention are heterodimers that contain constant heavy chainand/or constant light chain regions, and in particular, Fc domains. Thatis, some variants discussed herein are within the vhCH1, although manyof the variants are within the Fc domain (hinge-CH2-CH3).

As will be appreciated by those in the art and discussed more fullybelow, the heterodimeric fusion proteins of the present invention cantake on a wide variety of configurations, as are generally depicted inthe Figures. Some figures depict “single ended” configurations, wherethere is one type of specificity on one “arm” of the molecule and adifferent specificity on the other “arm”. Other figures depict “dualended” configurations, where there is at least one type of specificityat the “top” of the molecule and one or more different specificities atthe “bottom” of the molecule. Furthermore as is shown, these twoconfigurations can be combined, where there can be triple or quadruplespecificities based on the particular combination. Thus, the presentinvention provides “multispecific” binding proteins, includingmultispecific antibodies.

In some embodiments, the heterodimers resemble traditional antibodiesalthough they are bispecific and have two different variable regions;see FIG. 78. As outlined herein, the constant regions compriseheterodimerization variants, such as steric variants (“knobs in holes”,sometimes referred to in the art as “kih” variants) or pI variants, etc.In some cases, to reduce the complexity with regard to the light chains,some of these formats variable regions that share a common light chain(e.g. two separate heavy chains with a light chain that will assemblewith both but confers two different specificities.

In some embodiments, the heterodimers are bispecific in a formatgenerally referred to in the art as “CrossMab”. In this embodiment, inaddition to using the heterodimeric variants described herein, one heavychain monomer and one light chain monomer are also engineered such thatthe heavy chain monomer comprises a constant light region in place ofthe vhCH1domain, and the light chain contains the vhCH1 region with thevariable light region. This ensures that the correct light chains willpair with the correct heavy chains. See FIG. 78N and Schaefer et al.,PNAS 108(27) 11187-11192 (hereby incorporated by reference in itsentirety.

In some embodiments, sometimes referred to in the art as IgG-scFab, oneof the heavy chains has a scFab on it, such that one antigen is engagedbivalently and the other monovalently (e.g. two binding regions on one“end” and a single binding region on the other “end”). (See FIG. 78).

In some embodiments, sometimes referred to as mAb-Fv, each heavy chainof the heterodimer has an additional variable region on the terminus.One monomer has the variable heavy domain and the other monomer has avariable light domain (See FIG. 78E). See for example PCT US2010/047741,hereby incorporated by reference. In this embodiment, in general, thereare two different types of antibody analogs that allow for co-engagementmechanisms, one that utilizes three antigen binding domains (e.g. oneantigen is bound bivalently and the other is bound monovalently,although as is further described below, there can also be threedifferent antigens that are bound or a single antigen), and one thatrelies on two antigen binding domains (e.g. each antigen is boundmonovalently).

Additional Modifications

In addition to the modifications outlined above, other modifications canbe made. For example, the molecules may be stabilized by theincorporation of disulphide bridges linking the VH and VL domains(Reiter et al., 1996, Nature Biotech. 14:1239-1245, entirelyincorporated by reference). In addition, there are a variety of covalentmodifications of antibodies that can be made as outlined below.

Covalent modifications of antibodies are included within the scope ofthis invention, and are generally, but not always, donepost-translationally. For example, several types of covalentmodifications of the antibody are introduced into the molecule byreacting specific amino acid residues of the antibody with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesmay also be derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole and the like.

In addition, modifications at cysteines are particularly useful inantibody-drug conjugate (ADC) applications, further described below. Insome embodiments, the constant region of the antibodies can beengineered to contain one or more cysteines that are particularly “thiolreactive”, so as to allow more specific and controlled placement of thedrug moiety. See for example U.S. Pat. No. 7,521,541, incorporated byreference in its entirety herein.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing alpha-amino-containing residues includeimidoesters such as methyl picolinimidate; pyridoxal phosphate;pyridoxal; chloroborohydride; trinitrobenzenesulfonic acid;O-methylisourea; 2,4-pentanedione; and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to formO-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 1251 or 1311 to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R′—N═C═N—R′), where R and R′ are optionallydifferent alkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimide or 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide.Furthermore, aspartyl and glutamyl residues are converted to asparaginyland glutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinkingantibodies to a water-insoluble support matrix or surface for use in avariety of methods, in addition to methods described below. Commonlyused crosslinking agents include, e.g.,1,1-bis(diazoacetyl)-2-phenylethane, glutaraldehyde,N-hydroxysuccinimide esters, for example, esters with 4-azidosalicylicacid, homobifunctional imidoesters, including disuccinimidyl esters suchas 3,3′-dithiobis(succinimidylpropionate), and bifunctional maleimidessuch as bis-N-maleimido-1,8-octane. Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such ascynomolgusogen bromide-activated carbohydrates and the reactivesubstrates described in U.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128;4,247,642; 4,229,537; and 4,330,440, all entirely incorporated byreference, are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively.Alternatively, these residues are deamidated under mildly acidicconditions. Either form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983],entirely incorporated by reference), acetylation of the N-terminalamine, and amidation of any C-terminal carboxyl group.

In addition, as will be appreciated by those in the art, labels(including fluorescent, enzymatic, magnetic, radioactive, etc. can allbe added to the antibodies (as well as the other compositions of theinvention).

Glycosylation

Another type of covalent modification is alterations in glycosylation.In another embodiment, the antibodies disclosed herein can be modifiedto include one or more engineered glycoforms. By “engineered glycoform”as used herein is meant a carbohydrate composition that is covalentlyattached to the antibody, wherein said carbohydrate composition differschemically from that of a parent antibody. Engineered glycoforms may beuseful for a variety of purposes, including but not limited to enhancingor reducing effector function. A preferred form of engineered glycoformis afucosylation, which has been shown to be correlated to an increasein ADCC function, presumably through tighter binding to the FcγRIIIareceptor. In this context, “afucosylation” means that the majority ofthe antibody produced in the host cells is substantially devoid offucose, e.g. 90-95-98% of the generated antibodies do not haveappreciable fucose as a component of the carbohydrate moiety of theantibody (generally attached at N297 in the Fc region). Definedfunctionally, afucosylated antibodies generally exhibit at least a 50%or higher affinity to the FcγRIIIa receptor.

Engineered glycoforms may be generated by a variety of methods known inthe art (Umaña et al., 1999, Nat Biotechnol 17:176-180; Davies et al.,2001, Biotechnol Bioeng 74:288-294; Shields et al., 2002, J Biol Chem277:26733-26740; Shinkawa et al., 2003, J Biol Chem 278:3466-3473; U.S.Pat. No. 6,602,684; U.S. Ser. No. 10/277,370; U.S. Ser. No. 10/113,929;PCT WO 00/61739A1; PCT WO 01/29246A1; PCT WO 02/31140A1; PCT WO02/30954A1, all entirely incorporated by reference; (Potelligent®technology [Biowa, Inc., Princeton, N.J.]; GlycoMAb® glycosylationengineering technology [Glycart Biotechnology AG, Zurich, Switzerland]).Many of these techniques are based on controlling the level offucosylated and/or bisecting oligosaccharides that are covalentlyattached to the Fc region, for example by expressing an IgG in variousorganisms or cell lines, engineered or otherwise (for example Lec-13 CHOcells or rat hybridoma YB2/0 cells, by regulating enzymes involved inthe glycosylation pathway (for example FUT8 [α1,6-fucosyltranserase]and/or β1-4-N-acetylglucosaminyltransferase III [GnTIII]), or bymodifying carbohydrate(s) after the IgG has been expressed. For example,the “sugar engineered antibody” or “SEA technology” of Seattle Geneticsfunctions by adding modified saccharides that inhibit fucosylationduring production; see for example 20090317869, hereby incorporated byreference in its entirety. Engineered glycoform typically refers to thedifferent carbohydrate or oligosaccharide; thus an antibody can includean engineered glycoform.

Alternatively, engineered glycoform may refer to the IgG variant thatcomprises the different carbohydrate or oligosaccharide. As is known inthe art, glycosylation patterns can depend on both the sequence of theprotein (e.g., the presence or absence of particular glycosylation aminoacid residues, discussed below), or the host cell or organism in whichthe protein is produced. Particular expression systems are discussedbelow.

Glycosylation of polypeptides is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tri-peptide sequencesasparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tri-peptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose, to a hydroxyamino acid,most commonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to the antibody is convenientlyaccomplished by altering the amino acid sequence such that it containsone or more of the above-described tri-peptide sequences (for N-linkedglycosylation sites). The alteration may also be made by the additionof, or substitution by, one or more serine or threonine residues to thestarting sequence (for O-linked glycosylation sites). For ease, theantibody amino acid sequence is preferably altered through changes atthe DNA level, particularly by mutating the DNA encoding the targetpolypeptide at preselected bases such that codons are generated thatwill translate into the desired amino acids.

Another means of increasing the number of carbohydrate moieties on theantibody is by chemical or enzymatic coupling of glycosides to theprotein. These procedures are advantageous in that they do not requireproduction of the protein in a host cell that has glycosylationcapabilities for N- and O-linked glycosylation. Depending on thecoupling mode used, the sugar(s) may be attached to (a) arginine andhistidine, (b) free carboxyl groups, (c) free sulfhydryl groups such asthose of cysteine, (d) free hydroxyl groups such as those of serine,threonine, or hydroxyproline, (e) aromatic residues such as those ofphenylalanine, tyrosine, or tryptophan, or (f) the amide group ofglutamine. These methods are described in WO 87/05330 and in Aplin andWriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306, both entirelyincorporated by reference.

Removal of carbohydrate moieties present on the starting antibody (e.g.post-translationally) may be accomplished chemically or enzymatically.Chemical deglycosylation requires exposure of the protein to thecompound trifluoromethanesulfonic acid, or an equivalent compound. Thistreatment results in the cleavage of most or all sugars except thelinking sugar (N-acetylglucosamine or N-acetylgalactosamine), whileleaving the polypeptide intact. Chemical deglycosylation is described byHakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52 and by Edge etal., 1981, Anal. Biochem. 118:131, both entirely incorporated byreference. Enzymatic cleavage of carbohydrate moieties on polypeptidescan be achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., 1987, Meth. Enzymol. 138:350, entirelyincorporated by reference. Glycosylation at potential glycosylationsites may be prevented by the use of the compound tunicamycin asdescribed by Duskin et al., 1982, J. Biol. Chem. 257:3105, entirelyincorporated by reference. Tunicamycin blocks the formation ofprotein-N-glycoside linkages.

Another type of covalent modification of the antibody comprises linkingthe antibody to various nonproteinaceous polymers, including, but notlimited to, various polyols such as polyethylene glycol, polypropyleneglycol or polyoxyalkylenes, in the manner set forth in, for example,2005-2006 PEG Catalog from Nektar Therapeutics (available at the Nektarwebsite) U.S. Pat. Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417;4,791,192 or 4,179,337, all entirely incorporated by reference. Inaddition, as is known in the art, amino acid substitutions may be madein various positions within the antibody to facilitate the addition ofpolymers such as PEG. See for example, U.S. Publication No.2005/0114037A1, entirely incorporated by reference.

Other Fc Modifications

In addition to heterodimerization variants, other amino acidmodifications (particularly amino acid substitutions) find use to alteradditional properties of the heterodimer.

FcγR Variants

In one embodiment, the heterodimers of the invention can include aminoacid modifications to alter binding to one or more of the FcγRreceptors. Substitutions that result in increased binding as well asdecreased binding can be useful. For example, it is known that increasedbinding to FcγRIIIa generally results in increased ADCC (antibodydependent cell-mediated cytotoxicity; the cell-mediated reaction whereinnonspecific cytotoxic cells that express FcγRs recognize bound antibodyon a target cell and subsequently cause lysis of the target cell).Similarly, decreased binding to FcγRIIb (an inhibitory receptor) can bebeneficial as well in some circumstances Amino acid substitutions thatfind use in the present invention include those listed in U.S. Ser. No.11/124,620 (particularly FIG. 41, specifically incorporated herein),U.S. Ser. Nos. 11/174,287, 11/396,495, 11/538,406, all of which areexpressly incorporated herein by reference in their entirety andspecifically for the variants disclosed therein.

Particular variants that find use include, but are not limited to, 236A,239D, 239E, 332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F,236A/332E, 239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 243L,298A and 299T. Additional suitable Fc variants are found in FIG. 41 ofUS 2006/0024298, the figure and legend of which are hereby incorporatedby reference in their entirety.

FcRn Modifications

In addition, there are additional Fc substitutions that find use inincreased binding to the FcRn receptor and/or increased serum half life,as specifically disclosed in U.S. Ser. No. 12/341,769, herebyincorporated by reference in its entirety (particularly FIGS. 9 and 10),including, but not limited to, 434A, 434S, 428L, 308F, 259I, 428L/434S,259I/308F, 436I/428L, 436I or V/4345, 436V/428L, 252Y, 252Y/254T/256Eand 259I/308F/428L.

Binding Moieties/Targets

The heterodimeric proteins (for example the heterodimericimmunoglobulins) of the invention may target virtually any antigens. Asnoted above, there are a wide variety of suitable heterodimeric antibodyformats, with some preferably co-engage two target antigens, although insome cases, three or four antigens can be engaged.

Particular suitable applications of the immunoglobulins herein areco-target pairs for which it is beneficial or critical to engage atarget antigen monovalently. Such antigens may be, for example, immunereceptors that are activated upon immune complexation. Cellularactivation of many immune receptors occurs only by cross-linking,achieved typically by antibody/antigen immune complexes, or via effectorcell to target cell engagement. For some immune receptors, for examplethe CD3 signaling receptor on T cells, activation only upon engagementwith co-engaged target is critical, as nonspecific cross-linking in aclinical setting can elicit a cytokine storm and toxicity.Therapeutically, by engaging such antigens monovalently rather thanmultivalently, using the immunoglobulins herein, such activation occursonly in response to cross-linking only in the microenvironment of theprimary target antigen. The ability to target two different antigenswith different valencies is a novel and useful aspect of the presentinvention. Examples of target antigens for which it may betherapeutically beneficial or necessary to co-engage monovalentlyinclude but are not limited to immune activating receptors such as CD3,FcγRs, toll-like receptors (TLRs) such as TLR4 and TLR9, cytokine,chemokine, cytokine receptors, and chemokine receptors.

Virtually any antigen may be targeted by the immunoglobulins herein,including but not limited to proteins, subunits, domains, motifs, and/orepitopes belonging to the following list of target antigens, whichincludes both soluble factors such as cytokines and membrane-boundfactors, including transmembrane receptors: 17-IA, 4-1BB, 4Dc,6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 Adenosine Receptor, A33, ACE,ACE-2, Activin, Activin A, Activin AB, Activin B, Activin C, ActivinRIA, Activin RIA ALK-2, Activin RIB ALK-4, Activin RITA, Activin RIIB,ADAM, ADAM10, ADAM12, ADAM15, ADAM17/TACE, ADAMS, ADAMS, ADAMTS,ADAMTS4, ADAMTS5, Addressins, aFGF, ALCAM, ALK, ALK-1, ALK-7,alpha-1-antitrypsin, alpha-V/beta-1 antagonist, ANG, Ang, APAF-1, APE,APJ, APP, APRIL, AR, ARC, ART, Artemin, anti-Id, ASPARTIC, Atrialnatriuretic factor, av/b3 integrin, Ax1, b2M, B7-1, B7-2, B7-H,B-lymphocyte Stimulator (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1,BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM,BLC, BL-CAM, BLK, BMP, BMP-2 BMP-2a, BMP-3 Osteogenin, BMP-4 BMP-2b,BMP-5, BMP-6 Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA(ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF,BOK, Bombesin, Bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC,complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8,Calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associatedantigen, Cathepsin A, Cathepsin B, Cathepsin C/DPPI, Cathepsin D,Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S,Cathepsin V, Cathepsin X/Z/P, CBL, CCI, CCK2, CCL, CCL1, CCL11, CCL12,CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21,CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6,CCL7, CCL8, CCL9/10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5,CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8,CD10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20,CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33 (p67proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD49a, CD52, CD54,CD55, CD56, CD61, CD64, CD66e, CD74, CD80 (B7-1), CD89, CD95, CD123,CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR,cGMP, CINC, Clostridium botulinum toxin, Clostridium perfringens toxin,CKb8-1, CLC, CMV, CMV UL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK,CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5,CXCL6, CXCL7, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14,CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6,cytokeratin tumor-associated antigen, DAN, DCC, DcR3, DC-SIGN, Decayaccelerating factor, des(1-3)-IGF-I (brain IGF-1), Dhh, digoxin, DNAM-1,Dnase, Dpp, DPPIV/CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR(ErbB-1), EMA, EMMPRIN, ENA, endothelin receptor, Enkephalinase, eNOS,Eot, eotaxin1, EpCAM, Ephrin B2/EphB4, EPO, ERCC, E-selectin, ET-1,Factor Ha, Factor VII, Factor VIIIc, Factor IX, fibroblast activationprotein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3,FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Folliclestimulating hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6,FZD7, FZD8, FZD9, FZD10, G250, Gas 6, GCP-2, GCSF, GD2, GD3, GDF, GDF-1,GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7(BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF,GFAP, GFRa-1, GFR-alpha1, GFR-alpha2, GFR-alpha3, GITR, Glucagon, Glut4, glycoprotein IIb/IIIa (GP IIb/IIIa), GM-CSF, gp130, gp72, GRO, Growthhormone releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL,Hemopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gBglycoprotein, HSV gD glycoprotein, HGFA, High molecular weightmelanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp 120 V3loop, HLA, HLA-DR, HM1.24, HMFG PEM, HRG, Hrk, human cardiac myosin,human cytomegalovirus (HCMV), human growth hormone (HGH), HVEM, 1-309,IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF,IGF binding proteins, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R,IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10,IL-12, IL-13, IL-15, IL-18, IL-18R, IL-23, interferon (INF)-alpha,INF-beta, INF-gamma, Inhibin, iNOS, Insulin A-chain, Insulin B-chain,Insulin-like growth factor 1, integrin alpha2, integrin alpha3, integrinalpha4, integrin alpha4/beta1, integrin alpha4/beta7, integrin alpha5(alphaV), integrin alpha5/beta1, integrin alpha5/beta3, integrin alpha6,integrin beta1, integrin beta2, interferon gamma, IP-10, I-TAC, JE,Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12,Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, KallikreinL3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), laminin 5,LAMP, LAP, LAP (TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2,Lefty, Lewis-Y antigen, Lewis-Y related antigen, LFA-1, LFA-3, Lfo, LIF,LIGHT, lipoproteins, LIX, LKN, Lptn, L-Selectin, LT-a, LT-b, LTB4,LTBP-1, Lung surfactant, Luteinizing hormone, Lymphotoxin Beta Receptor,Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer,METALLOPROTEASES, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP,MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13,MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo,MSK, MSP, mucin (Muc1), MUC18, Muellerian-inhibitin substance, Mug,MuSK, NAIP, NAP, NCAD, N-Cadherin, NCA 90, NCAM, NCAM, Neprilysin,Neurotrophin-3, -4, or -6, Neurturin, Neuronal growth factor (NGF),NGFR, NGF-beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN,OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid hormone, PARC, PARP,PBR, PBSF, PCAD, P-Cadherin, PCNA, PDGF, PDGF, PDK-1, PECAM, PEM, PF4,PGE, PGF, PGI2, PGD2, PIN, PLA2, placental alkaline phosphatase (PLAP),P1GF, PLP, PP14, Proinsulin, Prorelaxin, Protein C, PS, PSA, PSCA,prostate specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51,RANK, RANKL, RANTES, RANTES, Relaxin A-chain, Relaxin B-chain, renin,respiratory syncytial virus (RSV) F, RSV Fgp, Ret, Rheumatoid factors,RLIP76, RPA2, RSK, S100, SCF/KL, SDF-1, SERINE, Serum albumin, sFRP-3,Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat,STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72),TARC, TCA-3, T-cell receptors (e.g., T-cell receptor alpha/beta), TdT,TECK, TEM1, TEM5, TEM7, TEM8, TERT, testicular PLAP-like alkalinephosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan Specific,TGF-beta RI (ALK-5), TGF-beta RII, TGF-beta RIIb, TGF-beta RIII,TGF-beta1, TGF-beta2, TGF-beta3, TGF-beta4, TGF-beta5, Thrombin, ThymusCk-1, Thyroid stimulating hormone, Tie, TIMP, TIQ, Tissue Factor,TMEFF2, Tmpo, TMPRSS2, TNF, TNF-alpha, TNF-alpha beta, TNF-beta2, TNFc,TNF-RI, TNF-RII, TNFRSF10A (TRAIL R1 Apo-2, DR4), TNFRSF10B (TRAIL R2DR5, KILLER, TRICK-2A, TRICK-B), TNFRSF10C (TRAIL R3 DcR1, LIT, TRID),TNFRSF10D (TRAIL R4 DcR2, TRUNDD), TNFRSF11A (RANK ODF R, TRANCE R),TNFRSF11B (OPG OCIF, TR1), TNFRSF12 (TWEAK R FN14), TNFRSF13B (TACT),TNFRSF13C (BAFF R), TNFRSF14 (HVEM ATAR, HveA, LIGHT R, TR2), TNFRSF 16(NGFR p75NTR), TNFRSF 17 (BCMA), TNFRSF 18 (GITR AITR), TNFRSF19 (TROYTAJ, TRADE), TNFRSF19L (RELT), TNFRSF1A (TNF RI CD120a, p55-60),TNFRSF1B (TNF RII CD120b, p75-80), TNFRSF26 (TNFRH3), TNFRSF3 (LTbR TNFRIII, TNFC R), TNFRSF4 (OX40 ACT35, TXGP1R), TNFRSF5 (CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B (DcR3 M68, TR6), TNFRSF7 (CD27),TNFRSF8 (CD30), TNFRSF9 (4-1BB CD137, ILA), TNFRSF21 (DR6), TNFRSF22(DcTRAIL R2 TNFRH2), TNFRST23 (DcTRAIL R1 TNFRH1), TNFRSF25 (DR3 Apo-3,LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL Apo-2 Ligand, TL2), TNFSF11(TRANCE/RANK Ligand ODF, OPG Ligand), TNFSF12 (TWEAK Apo-3 Ligand, DR3Ligand), TNFSF13 (APRIL TALL2), TNFSF13B (BAFF BLYS, TALL1, THANK,TNFSF20), TNFSF14 (LIGHT HVEM Ligand, LTg), TNFSF15 (TL1A/VEGI), TNFSF18(GITR Ligand AITR Ligand, TL6), TNFSF1A (TNF-a Conectin, DIF, TNFSF2),TNFSF1B (TNF-b LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 Ligandgp34, TXGP1), TNFSF5 (CD40 Ligand CD154, gp39, HIGM1, IMD3, TRAP),TNFSF6 (Fas Ligand Apo-1 Ligand, APT1 Ligand), TNFSF7 (CD27 LigandCD70), TNFSF8 (CD30 Ligand CD153), TNFSF9 (4-1BB Ligand CD137 Ligand),TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE,transferring receptor, TRF, Trk, TROP-2, TSG, TSLP, tumor-associatedantigen CA 125, tumor-associated antigen expressing Lewis Y relatedcarbohydrate, TWEAK, TXB2, Ung, uPAR, uPAR-1, Urokinase, VCAM, VCAM-1,VECAD, VE-Cadherin, VE-cadherin-2, VEFGR-1 (flt-1), VEGF, VEGFR, VEGFR-3(fit-4), VEGI, VIM, Viral antigens, VLA, VLA-1, VLA-4, VNR integrin, vonWillebrands factor, WIF-1, WNT1, WNT2, WNT2B/13, WNT3, WNT3A, WNT4,WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B,WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD,and receptors for hormones and growth factors. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

Exemplary antigens that may be targeted specifically by theimmunoglobulins of the invention include but are not limited to: CD20,CD19, Her2, EGFR, EpCAM, CD3, FcγRIIIa (CD16), FcγRIIa (CD32a), FcγRIIb(CD32b), FcγRI (CD64), Toll-like receptors (TLRs) such as TLR4 and TLR9,cytokines such as IL-2, IL-5, IL-13, IL-12, IL-23, and TNFα, cytokinereceptors such as IL-2R, chemokines, chemokine receptors, growth factorssuch as VEGF and HGF, and the like. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The choice of suitable target antigens and co-targets depends on thedesired therapeutic application. Some targets that have provenespecially amenable to antibody therapy are those with signalingfunctions. Other therapeutic antibodies exert their effects by blockingsignaling of the receptor by inhibiting the binding between a receptorand its cognate ligand. Another mechanism of action of therapeuticantibodies is to cause receptor down regulation. Other antibodies do notwork by signaling through their target antigen. The choice of co-targetswill depend on the detailed biology underlying the pathology of theindication that is being treated.

Monoclonal antibody therapy has emerged as an important therapeuticmodality for cancer (Weiner et al., 2010, Nature Reviews Immunology10:317-327; Reichert et al., 2005, Nature Biotechnology 23[9]:1073-1078;herein expressly incorporated by reference). For anti-cancer treatmentit may be desirable to target one antigen (antigen-1) whose expressionis restricted to the cancerous cells while co-targeting a second antigen(antigen-2) that mediates some immunological killing activity. For othertreatments it may be beneficial to co-target two antigens, for exampletwo angiogenic factors or two growth factors, that are each known toplay some role in proliferation of the tumor. Exemplary co-targets foroncology include but are not limited to HGF and VEGF, IGF-1R and VEGF,Her2 and VEGF, CD19 and CD3, CD20 and CD3, Her2 and CD3, CD19 andFcγRIIIa, CD20 and FcγRIIIa, Her2 and FcγRIIIa. An immunoglobulin of theinvention may be capable of binding VEGF and phosphatidylserine; VEGFand ErbB3; VEGF and PLGF; VEGF and ROBO4; VEGF and BSG2; VEGF and CDCP1;VEGF and ANPEP; VEGF and c-MET; HER-2 and ERB3; HER-2 and BSG2; HER-2and CDCP1; HER-2 and ANPEP; EGFR and CD64; EGFR and BSG2; EGFR andCDCP1; EGFR and ANPEP; IGF1R and PDGFR; IGF1R and VEGF; IGF1R and CD20;CD20 and CD74; CD20 and CD30; CD20 and DR4; CD20 and VEGFR2; CD20 andCD52; CD20 and CD4; HGF and c-MET; HGF and NRP1; HGF andphosphatidylserine; ErbB3 and IGF1R; ErbB3 and IGF1,2; c-Met and Her-2;c-Met and NRP1; c-Met and IGF1R; IGF1,2 and PDGFR; IGF1,2 and CD20;IGF1,2 and IGF1R; IGF2 and EGFR; IGF2 and HER2; IGF2 and CD20; IGF2 andVEGF; IGF2 and IGF1R; IGF1 and IGF2; PDGFRa and VEGFR2; PDGFRa and PLGF;PDGFRa and VEGF; PDGFRa and c-Met; PDGFRa and EGFR; PDGFRb and VEGFR2;PDGFRb and c-Met; PDGFRb and EGFR; RON and c-Met; RON and MTSP1; RON andMSP; RON and CDCP1; VGFR1 and PLGF; VGFR1 and RON; VGFR1 and EGFR;VEGFR2 and PLGF; VEGFR2 and NRP1; VEGFR2 and RON; VEGFR2 and DLL4;VEGFR2 and EGFR; VEGFR2 and ROBO4; VEGFR2 and CD55; LPA and S1P; EPHB2and RON; CTLA4 and VEGF; CD3 and EPCAM; CD40 and IL6; CD40 and IGF; CD40and CD56; CD40 and CD70; CD40 and VEGFR1; CD40 and DR5; CD40 and DR4;CD40 and APRIL; CD40 and BCMA; CD40 and RANKL; CD28 and MAPG; CD80 andCD40; CD80 and CD30; CD80 and CD33; CD80 and CD74; CD80 and CD2; CD80and CD3; CD80 and CD19; CD80 and CD4; CD80 and CD52; CD80 and VEGF; CD80and DR5; CD80 and VEGFR2; CD22 and CD20; CD22 and CD80; CD22 and CD40;CD22 and CD23; CD22 and CD33; CD22 and CD74; CD22 and CD19; CD22 andDR5; CD22 and DR4; CD22 and VEGF; CD22 and CD52; CD30 and CD20; CD30 andCD22; CD30 and CD23; CD30 and CD40; CD30 and VEGF; CD30 and CD74; CD30and CD19; CD30 and DR5; CD30 and DR4; CD30 and VEGFR2; CD30 and CD52;CD30 and CD4; CD138 and RANKL; CD33 and FTL3; CD33 and VEGF; CD33 andVEGFR2; CD33 and CD44; CD33 and DR4; CD33 and DR5; DR4 and CD137; DR4and IGF1,2; DR4 and IGF1R; DR4 and DR5; DR5 and CD40; DR5 and CD137; DR5and CD20; DR5 and EGFR; DR5 and IGF1,2; DR5 and IGFR, DR5 and HER-2, andEGFR and DLL4. Other target combinations include one or more members ofthe EGF/erb-2/erb-3 family.

Other targets (one or more) involved in oncological diseases that theimmunoglobulins herein may bind include, but are not limited to thoseselected from the group consisting of: CD52, CD20, CD19, CD3, CD4, CD8,BMP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, TNF, TNFSF10, BMP6, EGF, FGF1,FGF10, FGF11, FGF12, FGF13, FGF14, FGF16, FGF17, FGF18, FGF19, FGF2,FGF20, FGF21, FGF22, FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9,GRP, IGF1, IGF2, IL12A, IL1A, IL1B, 1L2, INHA, TGFA, TGFB1, TGFB2,TGFB3, VEGF, CDK2, FGF10, FGF18, FGF2, FGF4, FGF7, IGF1R, IL2, BCL2,CD164, CDKN1A, CDKN1B, CDKN1C, CDKN2A, CDKN2B, CDKN2C, CDKN3, GNRH1,IGFBP6, IL1A, IL1B, ODZ1, PAWR, PLG, TGFB1I1, AR, BRCA1, CDK3, CDK4,CDK5, CDK6, CDK7, CDK9, E2F1, EGFR, ENO1, ERBB2, ESR1, ESR2, IGFBP3,IGFBP6, IL2, INSL4, MYC, NOX5, NR6A1, PAP, PCNA, PRKCQ, PRKD1, PRL,TP53, FGF22, FGF23, FGF9, IGFBP3, IL2, INHA, KLK6, TP53, CHGB, GNRH1,IGF1, IGF2, INHA, INSL3, INSL4, PRL, KLK6, SHBG, NR1D1, NR1H3, NR1I3,NR2F6, NR4A3, ESR1, ESR2, NROB1, NROB2, NR1D2, NR1H2, NR1H4, NR112,NR2C1, NR2C2, NR2E1, NR2E3, NR2F1, NR2F2, NR3C1, NR3C2, NR4A1, NR4A2,NR5A1, NR5A2, NR6 μl, PGR, RARB, FGF1, FGF2, FGF6, KLK3, KRT1, APOC1,BRCA1, CHGA, CHGB, CLU, COL1A1, COL6A1, EGF, ERBB2, ERK8, FGF1, FGF10,FGF11, FGF13, FGF14, FGF16, FGF17, FGF18, FGF2, FGF20, FGF21, FGF22,FGF23, FGF3, FGF4, FGF5, FGF6, FGF7, FGF8, FGF9, GNRH1, IGF1, IGF2,IGFBP3, IGFBP6, IL12A, IL1A, IL1B, 1L2, IL24, INHA, INSL3, INSL4, KLK10,KLK12, KLK13, KLK14, KLK15, KLK3, KLK4, KLK5, KLK6, KLK9, MMP2, MMP9,MSMB, NTN4, ODZ1, PAP, PLAU, PRL, PSAP, SERPINA3, SHBG, TGFA, TIMP3,CD44, CDH1, CDH10, CDH19, CDH20, CDH7, CDH9, CDH1, CDH10, CDH13, CDH18,CDH19, CDH20, CDH7, CDH8, CDH9, ROBO2, CD44, ILK, ITGA1, APC, CD164,COL6A1, MTSS1, PAP, TGFB1I1, AGR2, AIG1, AKAP1, AKAP2, CANT1, CAV1,CDH12, CLDN3, CLN3, CYBS, CYC1, DAB21P, DES, DNCL1, ELAC2, ENO2, ENO3,FASN, FLJ12584, FLJ25530, GAGEB1, GAGEC1, GGT1, GSTP1, HIP 1, HUMCYT2A,IL29, K6HF, KAI1, KRT2A, MIB1, PART1, PATE, PCA3, PIAS2, PIK3CG, PPID,PR1, PSCA, SLC2A2, SLC33 μl, SLC43 μl, STEAP, STEAP2, TPM1, TPM2, TRPC6,ANGPT1, ANGPT2, ANPEP, ECGF1, EREG, FGF1, FGF2, FIGF, FLT1, JAG1, KDR,LAMAS, NRP1, NRP2, PGF, PLXDC1, STAB 1, VEGF, VEGFC, ANGPTL3, BAILCOL4A3, IL8, LAMAS, NRP1, NRP2, STAB 1, ANGPTL4, PECAM1, PF4, PROK2,SERPINF1, TNFAIP2, CCL11, CCL2, CXCL1, CXCL10, CXCL3, CXCL5, CXCL6,CXCL9, IFNA1, IFNB1, IFNG, IL1B, IL6, MDK, EDG1, EFNA1, EFNA3, EFNB2,EGF, EPHB4, FGFR3, HGF, IGF1, ITGB3, PDGFA, TEK, TGFA, TGFB1, TGFB2,TGFBR1, CCL2, CDH5, COL1A1, EDG1, ENG, ITGAV, ITGB3, THBS1, THBS2, BAD,BAG1, BCL2, CCNA1, CCNA2, CCND1, CCNE1, CCNE2, CDH1 (E-cadherin), CDKN1B(p27Kip1), CDKN2A (p161NK4a), COL6A1, CTNNB1 (b-catenin), CTSB(cathepsin B), ERBB2 (Her-2), ESR1, ESR2, F3 (TF), FOSL1 (FRA-1), GATA3,GSN (Gelsolin), IGFBP2, IL2RA, IL6, IL6R, IL6ST (glycoprotein 130),ITGA6 (a6 integrin), JUN, KLK5, KRT19, MAP2K7 (c-Jun), MKI67 (Ki-67),NGFB (GF), NGFR, NME1 (M23A), PGR, PLAU (uPA), PTEN, SERPINB5 (maspin),SERPINE1 (PAI-1), TGFA, THBS1 (thrombospondin-1), TIE (Tie-1), TNFRSF6(Fas), TNFSF6 (FasL), TOP2A (topoisomerase Iia), TP53, AZGP1(zinc-a-glycoprotein), BPAG1 (plectin), CDKN1A (p21Wap1/Cip1), CLDN7(claudin-7), CLU (clusterin), ERBB2 (Her-2), FGF1, FLRT1 (fibronectin),GABRP (GABAa), GNAS1, ID2, ITGA6 (a6 integrin), ITGB4 (b 4 integrin),KLF5 (GC Box BP), KRT19 (Keratin 19), KRTHB6 (hair-specific type IIkeratin), MACMARCKS, MT3 (metallothionectin-III), MUC1 (mucin), PTGS2(COX-2), RAC2 (p21Rac2), S100A2, SCGB1D2 (lipophilin B), SCGB2A1(mammaglobin 2), SCGB2A2 (mammaglobin 1), SPRR1B (Spr1), THBS1, THBS2,THBS4, and TNFAIP2 (B94), RON, c-Met, CD64, DLL4, PLGF, CTLA4,phophatidylserine, ROBO4, CD80, CD22, CD40, CD23, CD28, CD80, CD55,CD38, CD70, CD74, CD30, CD138, CD56, CD33, CD2, CD137, DR4, DR5, RANKL,VEGFR2, PDGFR, VEGFR1, MTSP1, MSP, EPHB2, EPHA1, EPHA2, EpCAM, PGE2,NKG2D, LPA, SIP, APRIL, BCMA, MAPG, FLT3, PDGFR alpha, PDGFR beta, ROR1,PSMA, PSCA, SCD1, and CD59. To form the bispecific or trispecificantibodies of the invention, antibodies to any combination of theseantigens can be made; that is, each of these antigens can be optionallyand independently included or excluded from a multispecific antibodyaccording to the present invention.

Monoclonal antibody therapy has become an important therapeutic modalityfor treating autoimmune and inflammatory disorders (Chan & Carter, 2010,Nature Reviews Immunology 10:301-316; Reichert et al., 2005, NatureBiotechnology 23[9]:1073-1078; herein expressly incorporated byreference). Many proteins have been implicated in general autoimmune andinflammatory responses, and thus may be targeted by the immunogloublinsof the invention. Autoimmune and inflammatory targets include but arenot limited to C5, CCL1 (I-309), CCL11 (eotaxin), CCL13 (mcp-4), CCL15(MIP-1d), CCL16 (HCC-4), CCL17 (TARC), CCL18 (PARC), CCL19, CCL2(mcp-1), CCL20 (MIP-3a), CCL21 (MIP-2), CCL23 (MPIF-1), CCL24(MPIF-2/eotaxin-2), CCL25 (TECK), CCL26, CCL3 (MIP-1a), CCL4 (MIP-1b),CCL5 (RANTES), CCL7 (mcp-3), CCL8 (mcp-2), CXCL1, CXCL10 (IP-10), CXCL11(1-TAC/IP-9), CXCL12 (SDF1), CXCL13, CXCL14, CXCL2, CXCL3, CXCL5(ENA-78/LIX), CXCL6 (GCP-2), CXCL9, IL13, IL8, CCL13 (mcp-4), CCR1,CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CR1, IL8RA, XCR1(CCXCR1), IFNA2, IL10, IL13, IL17C, IL1A, IL1B, IL1F10, IL1F5, IL1F6,IL1F7, IL1F8, IL1F9, IL22, IL5, IL8, IL9, LTA, LTB, MIF, SCYE1(endothelial Monocyte-activating cytokine), SPP1, TNF, TNFSF5, IFNA2,IL10RA, IL10RB, IL13, IL13RA1, IL5RA, IL9, IL9R, ABCF1, BCL6, C3, C4A,CEBPB, CRP, ICEBERG, IL1R1, IL1RN, IL8RB, LTB4R, TOLLIP, FADD, IRAK1,IRAK2, MYD88, NCK2, TNFAIP3, TRADD, TRAF1, TRAF2, TRAF3, TRAF4, TRAF5,TRAF6, ACVR1, ACVR1B, ACVR2, ACVR2B, ACVRL1, CD28, CD3E, CD3G, CD3Z,CD69, CD80, CD86, CNR1, CTLA4, CYSLTR1, FCER1A, FCER2, FCGR3A, GPR44,HAVCR2, OPRD1, P2RX7, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9,TLR10, BLR1, CCL1, CCL2, CCL3, CCL4, CCL5, CCL7, CCL8, CCL11, CCL13,CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24,CCL25, CCR1, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CX3CL1,CX3CR1, CXCL1, CXCL2, CXCL3, CXCL5, CXCL6, CXCL10, CXCL11, CXCL12,CXCL13, CXCR4, GPR2, SCYE1, SDF2, XCL1, XCL2, XCR1, AMH, AMHR2, BMPR1A,BMPR1B, BMPR2, C19orf10 (IL27w), CER1, CSF1, CSF2, CSF3, DKFZp451J0118,FGF2, GFI1, IFNA1, IFNB1, IFNG, IGF1, IL1A, IL1B, IL1R1, IL1R2, IL2,IL2RA, IL2RB, IL2RG, IL3, IL4, IL4R, IL5, IL5RA, IL6, IL6R, IL6ST, IL7,IL8, IL8RA, IL8RB, IL9, IL9R, IL10, IL10RA, IL10RB, IL11, IL12RA, IL12A,IL12B, IL12RB1, IL12RB2, IL13, IL13RA1, IL13RA2, IL15, IL15RA, IL16,IL17, IL17R, IL18, IL18R1, IL19, IL20, KITLG, LEP, LTA, LTB, LTB4R,LTB4R2, LTBR, MIF, NPPB, PDGFB, TBX21, TDGF1, TGFA, TGFB1, TGFB1I1,TGFB2, TGFB3, TGFB1, TGFBR1, TGFBR2, TGFBR3, TH1L, TNF, TNFRSF1A,TNFRSF1B, TNFRSF7, TNFRSF8, TNFRSF9, TNFRSF11A, TNFRSF21, TNFSF4,TNFSF5, TNFSF6, TNFSF11, VEGF, ZFPM2, and RNF110 (ZNF144). To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

Exemplary co-targets for autoimmune and inflammatory disorders includebut are not limited to IL-1 and TNFalpha, IL-6 and TNFalpha, IL-6 andIL-1, IgE and IL-13, IL-1 and IL-13, IL-4 and IL-13, IL-5 and IL-13,IL-9 and IL-13, CD19 and FcγRIIb, and CD79 and FcγRIIb.

Immunoglobulins of the invention with specificity for the followingpairs of targets to treat inflammatory disease are contemplated: TNF andIL-17A; TNF and RANKL; TNF and VEGF; TNF and SOST; TNF and DKK; TNF andalphaVbeta3; TNF and NGF; TNF and IL-23p19; TNF and IL-6; TNF and SOST;TNF and IL-6R; TNF and CD-20; IgE and IL-13; IL-13 and IL23p19; IgE andIL-4; IgE and IL-9; IgE and IL-9; IgE and IL-13; IL-13 and IL-9; IL-13and IL-4; IL-13 and IL-9; IL-13 and IL-9; IL-13 and IL-4; IL-13 andIL-23p19; IL-13 and IL-9; IL-6R and VEGF; IL-6R and IL-17A; IL-6R andRANKL; IL-17A and IL-1beta; IL-1beta and RANKL; IL-1beta and VEGF; RANKLand CD-20; IL-1alpha and IL-1beta; IL-1alpha and IL-1beta.

Pairs of targets that the immunoglobulins described herein can bind andbe useful to treat asthma may be determined. In an embodiment, suchtargets include, but are not limited to, IL-13 and IL-1beta, sinceIL-1beta is also implicated in inflammatory response in asthma; IL-13and cytokines and chemokines that are involved in inflammation, such asIL-13 and IL-9; IL-13 and IL-4; IL-13 and IL-5; IL-13 and IL-25; IL-13and TARC; IL-13 and MDC; IL-13 and MIF; IL-13 and TGF-β; IL-13 and LHRagonist; IL-13 and CL25; IL-13 and SPRR2a; IL-13 and SPRR2b; and IL-13and ADAMS. The immunoglobulins herein may have specificity for one ormore targets involved in asthma selected from the group consisting ofCSF1 (MCSF), CSF2 (GM-CSF), CSF3 (GCSF), FGF2, IFNA1, IFNB1, IFNG,histamine and histamine receptors, IL1A, IL1B, IL2, IL3, IL4, IL5, IL6,IL7, IL8, IL9, IL10, IL11, IL12A, IL12B, IL13, IL14, IL15, IL16, IL17,IL18, IL19, KITLG, PDGFB, IL2RA, IL4R, IL5RA, IL8RA, IL8RB, IL12RB1,IL12RB2, IL13RA1, IL13RA2, IL18R1, TSLP, CCLi, CCL2, CCL3, CCL4, CCL5,CCL7, CCL8, CCL13, CCL17, CCL18, CCL19, CCL20, CCL22, CCL24, CX3CL1,CXCL1, CXCL2, CXCL3, XCLi, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8,CX3CR1, GPR2, XCR1, FOS, GATA3, JAK1, JAK3, STATE, TBX21, TGFB1, TNF,TNFSF6, YY1, CYSLTR1, FCER1A, FCER2, LTB4R, TB4R2, LTBR, and Chitinase.To form the bispecific or trispecific antibodies of the invention,antibodies to any combination of these antigens can be made; that is,each of these antigens can be optionally and independently included orexcluded from a multispecific antibody according to the presentinvention.

Pairs of targets involved in rheumatoid arthritis (RA) may beco-targeted by the invention, including but not limited to TNF andIL-18; TNF and IL-12; TNF and IL-23; TNF and IL-1beta; TNF and MIF; TNFand IL-17; and TNF and IL-15.

Antigens that may be targeted in order to treat systemic lupuserythematosus (SLE) by the immunoglobulins herein include but are notlimited to CD-20, CD-22, CD-19, CD28, CD4, CD80, HLA-DRA, IL10, IL2,IL4, TNFRSF5, TNFRSF6, TNFSF5, TNFSF6, BLR1, HDAC4, HDAC5, HDAC7A,HDAC9, ICOSL, IGBP1, MS4A1, RGSI, SLA2, CD81, IFNB1, IL10, TNFRSF5,TNFRSF7, TNFSF5, AICDA, BLNK, GALNAC4S-6ST, HDAC4, HDAC5, HDAC7A, HDAC9,IL10, IL11, IL4, INHA, INHBA, KLF6, TNFRSF7, CD28, CD38, CD69, CD80,CD83, CD86, DPP4, FCER2, IL2RA, TNFRSF8, TNFSF7, CD24, CD37, CD40, CD72,CD74, CD79A, CD79B, CR2, ILIR2, ITGA2, ITGA3, MS4A1, ST6GALI, CDIC,CHSTIO, HLA-A, HLA-DRA, and NTSE; CTLA4, B7.1, B7.2, BlyS, BAFF, C5,IL-4, IL-6, IL-10, IFN-α, and TNF-α. To form the bispecific ortrispecific antibodies of the invention, antibodies to any combinationof these antigens can be made; that is, each of these antigens can beoptionally and independently included or excluded from a multispecificantibody according to the present invention.

The immunoglobulins herein may target antigens for the treatment ofmultiple sclerosis (MS), including but not limited to IL-12, TWEAK,IL-23, CXCL13, CD40, CD40L, IL-18, VEGF, VLA-4, TNF, CD45RB, CD200,IFNgamma, GM-CSF, FGF, C5, CD52, and CCR2. An embodiment includesco-engagement of anti-IL-12 and TWEAK for the treatment of MS.

One aspect of the invention pertains to immunoglobulins capable ofbinding one or more targets involved in sepsis, in an embodiment twotargets, selected from the group consisting TNF, IL-1, MIF, IL-6, IL-8,IL-18, IL-12, IL-23, FasL, LPS, Toll-like receptors, TLR-4, tissuefactor, MIP-2, ADORA2A, CASP1, CASP4, IL-10, IL-1B, NFκB1, PROC,TNFRSFIA, CSF3, CCR3, ILIRN, MIF, NFκB1, PTAFR, TLR2, TLR4, GPR44,HMOX1, midkine, IRAK1, NFκB2, SERPINAL SERPINEL and TREM1. To form thebispecific or trispecific antibodies of the invention, antibodies to anycombination of these antigens can be made; that is, each of theseantigens can be optionally and independently included or excluded from amultispecific antibody according to the present invention.

In some cases, immunoglobulins herein may be directed against antigensfor the treatment of infectious diseases.

Antibodies for Engineering

In some embodiments, the heterodimeric engineering and multispecificengineering described herein is done with portions of therapeuticantibodies. A number of antibodies that are approved for use, inclinical trials, or in development may benefit from the pI variants ofthe present invention. These antibodies are herein referred to as“clinical products and candidates”. Thus in a preferred embodiment, theheterodimerization variants may find use in a range of clinical productsand candidates. For example the heterodimerization variants of thepresent invention may find use in an antibody that has components, e.g.the variable domains, the CDRs, etc., of clinical antibodies including,but not limited to, rituximab (Rituxan®, IDEC/Genentech/Roche) (see forexample U.S. Pat. No. 5,736,137), a chimeric anti-CD20 antibody approvedto treat Non-Hodgkin's lymphoma; HuMax-CD20, an anti-CD20 currentlybeing developed by Genmab, an anti-CD20 antibody described in U.S. Pat.No. 5,500,362, AME-133 (Applied Molecular Evolution), hA20(Immunomedics, Inc.), HumaLYM (Intracel), and PRO70769(PCT/US2003/040426, entitled “Immunoglobulin Variants and UsesThereof”). A number of antibodies that target members of the family ofepidermal growth factor receptors, including EGFR (ErbB-1), Her2/neu(ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), may benefit from pI engineeredconstant region(s) of the invention. For example the pI engineeredconstant region(s) of the invention may find use in an antibody that issubstantially similar to trastuzumab (Herceptin®, Genentech) (see forexample U.S. Pat. No. 5,677,171), a humanized anti-Her2/neu antibodyapproved to treat breast cancer; pertuzumab (rhuMab-2C4, Omnitarg™),currently being developed by Genentech; an anti-Her2 antibody describedin U.S. Pat. No. 4,753,894; cetuximab (Erbitux®, Imclone) (U.S. Pat. No.4,943,533; PCT WO 96/40210), a chimeric anti-EGFR antibody in clinicaltrials for a variety of cancers; ABX-EGF (U.S. Pat. No. 6,235,883),currently being developed by Abgenix-Immunex-Amgen; HuMax-EGFr (U.S.Ser. No. 10/172,317), currently being developed by Genmab; 425,EMD55900, EMD62000, and EMD72000 (Merck KGaA) (U.S. Pat. No. 5,558,864;Murthy et al. 1987, Arch Biochem Biophys. 252(2):549-60; Rodeck et al.,1987, J Cell Biochem. 35(4):315-20; Kettleborough et al., 1991, ProteinEng. 4(7):773-83); ICR62 (Institute of Cancer Research) (PCT WO95/20045; Modjtahedi et al., 1993, J. Cell Biophys. 1993,22(1-3):129-46; Modjtahedi et al., 1993, Br J Cancer. 1993,67(2):247-53; Modjtahedi et al, 1996, Br J Cancer, 73(2):228-35;Modjtahedi et al, 2003, Int J Cancer, 105(2):273-80); TheraCIM hR3 (YMBiosciences, Canada and Centro de Immunologia Molecular, Cuba (U.S. Pat.No. 5,891,996; U.S. Pat. No. 6,506,883; Mateo et al, 1997,Immunotechnology, 3(1):71-81); mAb-806 (Ludwig Institute for CancerResearch, Memorial Sloan-Kettering) (Jungbluth et al. 2003, Proc NatlAcad Sci USA. 100(2):639-44); KSB-102 (KS Biomedix); MR1-1 (WAX,National Cancer Institute) (PCT WO 0162931A2); and SC100 (Scancell) (PCTWO 01/88138). In another preferred embodiment, the pI engineeredconstant region(s) of the present invention may find use in alemtuzumab(Campath®, Millenium), a humanized monoclonal antibody currentlyapproved for treatment of B-cell chronic lymphocytic leukemia. The pIengineered constant region(s) of the present invention may find use in avariety of antibodies that are substantially similar to other clinicalproducts and candidates, including but not limited to muromonab-CD3(Orthoclone OKT3®), an anti-CD3 antibody developed by OrthoBiotech/Johnson & Johnson, ibritumomab tiuxetan (Zevalin®), an anti-CD20antibody developed by IDEC/Schering AG, gemtuzumab ozogamicin(Mylotarg®), an anti-CD33 (p67 protein) antibody developed byCelltech/Wyeth, alefacept (Amevive®), an anti-LFA-3 Fc fusion developedby Biogen), abciximab (ReoPro®), developed by Centocor/Lilly,basiliximab (Simulect®), developed by Novartis, palivizumab (Synagis®),developed by MedImmune, infliximab (Remicade®), an anti-TNFalphaantibody developed by Centocor, adalimumab (Humira®), an anti-TNFalphaantibody developed by Abbott, Humicade™, an anti-TNFalpha antibodydeveloped by Celltech, etanercept (Enbrel®), an anti-TNFalpha Fc fusiondeveloped by Immunex/Amgen, ABX-CBL, an anti-CD147 antibody beingdeveloped by Abgenix, ABX-IL8, an anti-IL8 antibody being developed byAbgenix, ABX-MA1, an anti-MUC18 antibody being developed by Abgenix,Pemtumomab (R1549, 90Y-muHMFG1), an anti-MUC1 In development byAntisoma, Therex (R1550), an anti-MUC1 antibody being developed byAntisoma, AngioMab (AS1405), being developed by Antisoma, HuBC-1, beingdeveloped by Antisoma, Thioplatin (AS1407) being developed by Antisoma,Antegren® (natalizumab), an anti-alpha-4-beta-1 (VLA-4) andalpha-4-beta-7 antibody being developed by Biogen, VLA-1 mAb, ananti-VLA-1 integrin antibody being developed by Biogen, LTBR mAb, ananti-lymphotoxin beta receptor (LTBR) antibody being developed byBiogen, CAT-152, an anti-TGF-β2 antibody being developed by CambridgeAntibody Technology, J695, an anti-IL-12 antibody being developed byCambridge Antibody Technology and Abbott, CAT-192, an anti-TGFβ1antibody being developed by Cambridge Antibody Technology and Genzyme,CAT-213, an anti-Eotaxin1 antibody being developed by Cambridge AntibodyTechnology, LymphoStat-B™ an anti-Blys antibody being developed byCambridge Antibody Technology and Human Genome Sciences Inc.,TRAIL-R1mAb, an anti-TRAIL-R1 antibody being developed by CambridgeAntibody Technology and Human Genome Sciences, Inc., Avastin™(bevacizumab, rhuMAb-VEGF), an anti-VEGF antibody being developed byGenentech, an anti-HER receptor family antibody being developed byGenentech, Anti-Tissue Factor (ATF), an anti-Tissue Factor antibodybeing developed by Genentech, Xolair™ (Omalizumab), an anti-IgE antibodybeing developed by Genentech, Raptiva™ (Efalizumab), an anti-CD11aantibody being developed by Genentech and Xoma, MLN-02 Antibody(formerly LDP-02), being developed by Genentech and MilleniumPharmaceuticals, HuMax CD4, an anti-CD4 antibody being developed byGenmab, HuMax-IL15, an anti-IL15 antibody being developed by Genmab andAmgen, HuMax-Inflam, being developed by Genmab and Medarex,HuMax-Cancer, an anti-Heparanase I antibody being developed by Genmaband Medarex and Oxford GcoSciences, HuMax-Lymphoma, being developed byGenmab and Amgen, HuMax-TAC, being developed by Genmab, IDEC-131, andanti-CD40L antibody being developed by IDEC Pharmaceuticals, IDEC-151(Clenoliximab), an anti-CD4 antibody being developed by IDECPharmaceuticals, IDEC-114, an anti-CD80 antibody being developed by IDECPharmaceuticals, IDEC-152, an anti-CD23 being developed by IDECPharmaceuticals, anti-macrophage migration factor (MIF) antibodies beingdeveloped by IDEC Pharmaceuticals, BEC2, an anti-idiotypic antibodybeing developed by Imclone, IMC-1C11, an anti-KDR antibody beingdeveloped by Imclone, DC101, an anti-flk-1 antibody being developed byImclone, anti-VE cadherin antibodies being developed by Imclone,CEA-Cide™ (labetuzumab), an anti-carcinoembryonic antigen (CEA) antibodybeing developed by Immunomedics, LymphoCide™ (Epratuzumab), an anti-CD22antibody being developed by Immunomedics, AFP-Cide, being developed byImmunomedics, MyelomaCide, being developed by Immunomedics, LkoCide,being developed by Immunomedics, ProstaCide, being developed byImmunomedics, MDX-010, an anti-CTLA4 antibody being developed byMedarex, MDX-060, an anti-CD30 antibody being developed by Medarex,MDX-070 being developed by Medarex, MDX-018 being developed by Medarex,Osidem™ (IDM-1), and anti-Her2 antibody being developed by Medarex andImmuno-Designed Molecules, HuMax™-CD4, an anti-CD4 antibody beingdeveloped by Medarex and Genmab, HuMax-IL15, an anti-IL15 antibody beingdeveloped by Medarex and Genmab, CNTO 148, an anti-TNFα antibody beingdeveloped by Medarex and Centocor/J&J, CNTO 1275, an anti-cytokineantibody being developed by Centocor/J&J, MOR101 and MOR102,anti-intercellular adhesion molecule-1 (ICAM-1) (CD54) antibodies beingdeveloped by MorphoSys, MOR201, an anti-fibroblast growth factorreceptor 3 (FGFR-3) antibody being developed by MorphoSys, Nuvion®(visilizumab), an anti-CD3 antibody being developed by Protein DesignLabs, HuZAF™, an anti-gamma interferon antibody being developed byProtein Design Labs, Anti-α5β1 Integrin, being developed by ProteinDesign Labs, anti-IL-12, being developed by Protein Design Labs, ING-1,an anti-EpCAM antibody being developed by Xoma, and MLN01, an anti-Beta2integrin antibody being developed by Xoma, an pI-ADC antibody beingdeveloped by Seattle Genetics, all of the above-cited references in thisparagraph are expressly incorporated herein by reference.

The antibodies of the present invention are generally isolated orrecombinant. “Isolated,” when used to describe the various polypeptidesdisclosed herein, means a polypeptide that has been identified andseparated and/or recovered from a cell or cell culture from which it wasexpressed. Ordinarily, an isolated polypeptide will be prepared by atleast one purification step. An “isolated antibody,” refers to anantibody which is substantially free of other antibodies havingdifferent antigenic specificities.

“Specific binding” or “specifically binds to” or is “specific for” aparticular antigen or an epitope means binding that is measurablydifferent from a non-specific interaction. Specific binding can bemeasured, for example, by determining binding of a molecule compared tobinding of a control molecule, which generally is a molecule of similarstructure that does not have binding activity. For example, specificbinding can be determined by competition with a control molecule that issimilar to the target.

Specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KD for an antigen orepitope of at least about 10-4 M, at least about 10-5 M, at least about10-6 M, at least about 10-7 M, at least about 10-8 M, at least about10-9 M, alternatively at least about 10-10 M, at least about 10-11 M, atleast about 10-12 M, or greater, where KD refers to a dissociation rateof a particular antibody-antigen interaction. Typically, an antibodythat specifically binds an antigen will have a KD that is 20-, 50-,100-, 500-, 1000-, 5,000-, 10,000- or more times greater for a controlmolecule relative to the antigen or epitope.

Also, specific binding for a particular antigen or an epitope can beexhibited, for example, by an antibody having a KA or Ka for an antigenor epitope of at least 20-, 50-, 100-, 500-, 1000-, 5,000-, 10,000- ormore times greater for the epitope relative to a control, where KA or Karefers to an association rate of a particular antibody-antigeninteraction.

Methods for Making Heterodimers

As will be appreciated by those in the art, general techniques are usedto make and then purify the heterodimers as discussed herein and shownin the examples below.

As will be appreciated by those in the art, standard protocols are usedto make the multispecific binding proteins of the invention. Generalmethods for antibody molecular biology, expression, purification, andscreening are described in Antibody Engineering, edited by Kontermann &Dubel, Springer, Heidelberg, 2001; and Hayhurst & Georgiou, 2001, CurrOpin Chem Biol 5:683-689; Maynard & Georgiou, 2000, Annu Rev Biomed Eng2:339-76.

In one embodiment disclosed herein, nucleic acids are created thatencode the multispecific binding proteins, and that may then be clonedinto host cells, expressed and assayed, if desired. Thus, nucleic acids,and particularly DNA, may be made that encode each protein sequence.These practices are carried out using well-known procedures. Forexample, a variety of methods that may find use in generatingmultispecific binding proteins, similar to the production of antibodies,are disclosed herein are described in Molecular Cloning—A LaboratoryManual, 3rd Ed. (Maniatis, Cold Spring Harbor Laboratory Press, NewYork, 2001), and Current Protocols in Molecular Biology (John Wiley &Sons), both incorporated entirely by reference. There are a variety oftechniques that may be used to efficiently generate DNA encodingmultispecific binding proteins disclosed herein. Such methods includebut are not limited to gene assembly methods, PCR-based method andmethods which use variations of PCR, ligase chain reaction-basedmethods, pooled oligo methods such as those used in synthetic shuffling,error-prone amplification methods and methods which use oligos withrandom mutations, classical site-directed mutagenesis methods, cassettemutagenesis, and other amplification and gene synthesis methods. As isknown in the art, there are a variety of commercially available kits andmethods for gene assembly, mutagenesis, vector subcloning, and the like,and such commercial products find use in for generating nucleic acidsthat encode multispecific binding proteins.

The multispecific binding proteins disclosed herein may be produced byculturing a host cell transformed with nucleic acid, e.g., an expressionvector, containing nucleic acid encoding the multispecific bindingproteins, under the appropriate conditions to induce or cause expressionof the protein. The conditions appropriate for expression will vary withthe choice of the expression vector and the host cell, and will beeasily ascertained by one skilled in the art through routineexperimentation. A wide variety of appropriate host cells may be used,including but not limited to mammalian cells, bacteria, insect cells,yeast, and plant cells. For example, a variety of cell lines that mayfind use in generating multispecific binding proteins disclosed hereinare described in the ATCC® cell line catalog, available from theAmerican Type Culture Collection.

In one embodiment, the multispecific binding proteins are expressed inmammalian expression systems, including systems in which the expressionconstructs are introduced into the mammalian cells using virus such asretrovirus or adenovirus. Any mammalian cells may be used, e.g., human,mouse, rat, hamster, and primate cells. Suitable cells also includeknown research cells, including but not limited to Jurkat T cells,NIH3T3, CHO, BHK, COS, HEK293, PER C.6, HeLa, Sp2/0, NS0 cells andvariants thereof. In an alternate embodiment, library proteins areexpressed in bacterial cells. Bacterial expression systems are wellknown in the art, and include Escherichia coli (E. coli), Bacillussubtilis, Streptococcus cremoris, and Streptococcus lividans. Inalternate embodiments, antibodies are produced in insect cells (e.g.Sf21/5f9, Trichoplusia ni Bti-Tn5b1-4) or yeast cells (e.g. S.cerevisiae, Pichia, etc). In an alternate embodiment, antibodies areexpressed in vitro using cell free translation systems. In vitrotranslation systems derived from both prokaryotic (e.g. E. coli) andeukaryotic (e.g. wheat germ, rabbit reticulocytes) cells are availableand may be chosen based on the expression levels and functionalproperties of the protein of interest. For example, as appreciated bythose skilled in the art, in vitro translation is required for somedisplay technologies, for example ribosome display. In addition, theantibodies may be produced by chemical synthesis methods. Alsotransgenic expression systems both animal (e.g. cow, sheep or goat milk,embryonated hen's eggs, whole insect larvae, etc.) and plant (e.g. corn,tobacco, duckweed, etc.)

The nucleic acids that encode multispecific binding proteins disclosedherein may be incorporated into an expression vector in order to expressthe protein. A variety of expression vectors may be utilized for proteinexpression. Expression vectors may comprise self-replicatingextra-chromosomal vectors or vectors which integrate into a host genome.Expression vectors are constructed to be compatible with the host celltype. Thus expression vectors which find use in generating antibodiesdisclosed herein include but are not limited to those which enableprotein expression in mammalian cells, bacteria, insect cells, yeast,and in in vitro systems. As is known in the art, a variety of expressionvectors are available, commercially or otherwise, that may find use forexpressing antibodies disclosed herein.

The disclosed multispecific binding proteins can be encoded by multiplenucleic acid molecules. For example, the heavy and light chains of anantibody can be introduced into a host cell independently. Thoughpresent on separate nucleic acids, their expression yields a singlepolypeptide.

Expression vectors typically comprise a protein operably linked withcontrol or regulatory sequences, selectable markers, any fusionpartners, and/or additional elements. By “operably linked” herein ismeant that the nucleic acid is placed into a functional relationshipwith another nucleic acid sequence. Generally, these expression vectorsinclude transcriptional and translational regulatory nucleic acidoperably linked to the nucleic acid encoding the antibody, and aretypically appropriate to the host cell used to express the protein. Ingeneral, the transcriptional and translational regulatory sequences mayinclude promoter sequences, ribosomal binding sites, transcriptionalstart and stop sequences, translational start and stop sequences, andenhancer or activator sequences. As is also known in the art, expressionvectors typically contain a selection gene or marker to allow theselection of transformed host cells containing the expression vector.Selection genes are well known in the art and will vary with the hostcell used.

In one embodiment, multispecific binding proteins are purified orisolated after expression. Proteins may be isolated or purified in avariety of ways known to those skilled in the art. Purification may beparticularly useful in the invention for separating heterodimeric heavychain species from homodimeric heavy chain species, as described herein.Standard purification methods include chromatographic techniques,including ion exchange, hydrophobic interaction, affinity, sizing or gelfiltration, and reversed-phase, carried out at atmospheric pressure orat high pressure using systems such as FPLC and HPLC. Purificationmethods also include electrophoretic, isoelectric focusing,immunological, precipitation, dialysis, and chromatofocusing techniques.Ultrafiltration and diafiltration techniques, in conjunction withprotein concentration, are also useful. fusion is employed, Ni+2affinity chromatography if a His-tag is employed, or immobilizedanti-flag antibody if a flag-tag is used. For general guidance insuitable purification techniques, see, e.g. incorporated entirely byreference Protein Purification: Principles and Practice, 3rd Ed.,Scopes, Springer-Verlag, NY, 1994, incorporated entirely by reference.The degree of purification necessary will vary depending on the screenor use of the antibodies. In some instances no purification is needed.

Antibody-Drug Conjugates

In some embodiments, the multispecific antibodies of the invention areconjugated with drugs to form antibody-drug conjugates (ADCs). Ingeneral, ADCs are used in oncology applications, where the use ofantibody-drug conjugates for the local delivery of cytotoxic orcytostatic agents allows for the targeted delivery of the drug moiety totumors, which can allow higher efficacy, lower toxicity, etc. Anoverview of this technology is provided in Ducry et al., BioconjugateChem., 21:5-13 (2010), Carter et al., Cancer J. 14(3):154 (2008) andSenter, Current Opin. Chem. Biol. 13:235-244 (2009), all of which arehereby incorporated by reference in their entirety.

Thus the invention provides multispecific antibodies conjugated todrugs. Generally, conjugation is done by covalent attachment to theantibody, as further described below, and generally relies on a linker,often a peptide linkage (which, as described below, may be designed tobe sensitive to cleavage by proteases at the target site or not). Inaddition, as described above, linkage of the linker-drug unit (LU-D) canbe done by attachment to cysteines within the antibody. As will beappreciated by those in the art, the number of drug moieties perantibody can change, depending on the conditions of the reaction, andcan vary from 1:1 to 10:1 drug:antibody. As will be appreciated by thosein the art, the actual number is an average.

Thus the invention provides multispecific antibodies conjugated todrugs. As described below, the drug of the ADC can be any number ofagents, including but not limited to cytotoxic agents such aschemotherapeutic agents, growth inhibitory agents, toxins (for example,an enzymatically active toxin of bacterial, fungal, plant, or animalorigin, or fragments thereof), or a radioactive isotope (that is, aradioconjugate) are provided. In other embodiments, the inventionfurther provides methods of using the ADCs.

Drugs for use in the present invention include cytotoxic drugs,particularly those which are used for cancer therapy. Such drugsinclude, in general, DNA damaging agents, anti-metabolites, naturalproducts and their analogs. Exemplary classes of cytotoxic agentsinclude the enzyme inhibitors such as dihydrofolate reductaseinhibitors, and thymidylate synthase inhibitors, DNA intercalators, DNAcleavers, topoisomerase inhibitors, the anthracycline family of drugs,the vinca drugs, the mitomycins, the bleomycins, the cytotoxicnucleosides, the pteridine family of drugs, diynenes, thepodophyllotoxins, dolastatins, maytansinoids, differentiation inducers,and taxols.

Members of these classes include, for example, methotrexate,methopterin, dichloromethotrexate, 5-fluorouracil, 6-mercaptopurine,cytosine arabinoside, melphalan, leurosine, leurosideine, actinomycin,daunorubicin, doxorubicin, mitomycin C, mitomycin A, caminomycin,aminopterin, tallysomycin, podophyllotoxin and podophyllotoxinderivatives such as etoposide or etoposide phosphate, vinblastine,vincristine, vindesine, taxanes including taxol, taxotere retinoic acid,butyric acid, N8-acetyl spermidine, camptothecin, calicheamicin,esperamicin, ene-diynes, duocarmycin A, duocarmycin SA, calicheamicin,camptothecin, maytansinoids (including DM1), monomethylauristatin E(MMAE), monomethylauristatin F (MMAF), and maytansinoids (DM4) and theiranalogues.

Toxins may be used as antibody-toxin conjugates and include bacterialtoxins such as diphtheria toxin, plant toxins such as ricin, smallmolecule toxins such as geldanamycin (Mandler et al (2000) J. Nat.Cancer Inst. 92(19):1573-1581; Mandler et al (2000) Bioorganic & Med.Chem. Letters 10:1025-1028; Mandler et al (2002) Bioconjugate Chem.13:786-791), maytansinoids (EP 1391213; Liu et al., (1996) Proc. Natl.Acad. Sci. USA 93:8618-8623), and calicheamicin (Lode et al (1998)Cancer Res. 58:2928; Hinman et al (1993) Cancer Res. 53:3336-3342).Toxins may exert their cytotoxic and cytostatic effects by mechanismsincluding tubulin binding, DNA binding, or topoisomerase inhibition.

Conjugates of a multispecific antibody and one or more small moleculetoxins, such as a maytansinoids, dolastatins, auristatins, atrichothecene, calicheamicin, and CC1065, and the derivatives of thesetoxins that have toxin activity, are contemplated.

Maytansinoids

Maytansine compounds suitable for use as maytansinoid drug moieties arewell known in the art, and can be isolated from natural sourcesaccording to known methods, produced using genetic engineeringtechniques (see Yu et al (2002) PNAS 99:7968-7973), or maytansinol andmaytansinol analogues prepared synthetically according to known methods.As described below, drugs may be modified by the incorporation of afunctionally active group such as a thiol or amine group for conjugationto the antibody.

Exemplary maytansinoid drug moieties include those having a modifiedaromatic ring, such as: C-19-dechloro (U.S. Pat. No. 4,256,746)(prepared by lithium aluminum hydride reduction of ansamytocin P2);C-20-hydroxy (or C-20-demethyl)+/−C-19-dechloro (U.S. Pat. Nos.4,361,650 and 4,307,016) (prepared by demethylation using Streptomycesor Actinomyces or dechlorination using LAH); and C-20-demethoxy,C-20-acyloxy (—OCOR), +/− dechloro (U.S. Pat. No. 4,294,757) (preparedby acylation using acyl chlorides) and those having modifications atother positions

Exemplary maytansinoid drug moieties also include those havingmodifications such as: C-9-SH (U.S. Pat. No. 4,424,219) (prepared by thereaction of maytansinol with H2S or P2S5);C-14-alkoxymethyl(demethoxy/CH2OR) (U.S. Pat. No. 4,331,598);C-14-hydroxymethyl or acyloxymethyl (CH2OH or CH2OAc) (U.S. Pat. No.4,450,254) (prepared from Nocardia); C-15-hydroxy/acyloxy (U.S. Pat. No.4,364,866) (prepared by the conversion of maytansinol by Streptomyces);C-15-methoxy (U.S. Pat. Nos. 4,313,946 and 4,315,929) (isolated fromTrewia nudlflora); C-18-N-demethyl (U.S. Pat. Nos. 4,362,663 and4,322,348) (prepared by the demethylation of maytansinol byStreptomyces); and 4,5-deoxy (U.S. Pat. No. 4,371,533) (prepared by thetitanium trichloride/LAH reduction of maytansinol).

Of particular use are DM1 (disclosed in U.S. Pat. No. 5,208,020,incorporated by reference) and DM4 (disclosed in U.S. Pat. No.7,276,497, incorporated by reference). See also a number of additionalmaytansinoid derivatives and methods in 5,416,064, WO/01/24763,7,303,749, 7,601,354, U.S. Ser. No. 12/631,508, WO02/098883, 6,441,163,7,368,565, WO02/16368 and WO04/1033272, all of which are expresslyincorporated by reference in their entirety.

ADCs containing maytansinoids, methods of making same, and theirtherapeutic use are disclosed, for example, in U.S. Pat. Nos. 5,208,020;5,416,064; 6,441,163 and European Patent EP 0 425 235 B1, thedisclosures of which are hereby expressly incorporated by reference. Liuet al., Proc. Natl. Acad. Sci. USA 93:8618-8623 (1996) described ADCscomprising a maytansinoid designated DM1 linked to the monoclonalantibody C242 directed against human colorectal cancer. The conjugatewas found to be highly cytotoxic towards cultured colon cancer cells,and showed antitumor activity in an in vivo tumor growth assay.

Chari et al., Cancer Research 52:127-131 (1992) describe ADCs in which amaytansinoid was conjugated via a disulfide linker to the murineantibody A7 binding to an antigen on human colon cancer cell lines, orto another murine monoclonal antibody TA.1 that binds the HER-2/neuoncogene. The cytotoxicity of the TA.1-maytansonoid conjugate was testedin vitro on the human breast cancer cell line SK-BR-3, which expresses3×105 HER-2 surface antigens per cell. The drug conjugate achieved adegree of cytotoxicity similar to the free maytansinoid drug, whichcould be increased by increasing the number of maytansinoid moleculesper antibody molecule. The A7-maytansinoid conjugate showed low systemiccytotoxicity in mice.

Auristatins and Dolastatins

In some embodiments, the ADC comprises a multispecific antibodyconjugated to dolastatins or dolostatin peptidic analogs andderivatives, the auristatins (U.S. Pat. Nos. 5,635,483; 5,780,588).Dolastatins and auristatins have been shown to interfere withmicrotubule dynamics, GTP hydrolysis, and nuclear and cellular division(Woyke et al (2001) Antimicrob. Agents and Chemother. 45(12):3580-3584)and have anticancer (U.S. Pat. No. 5,663,149) and antifungal activity(Pettit et al (1998) Antimicrob. Agents Chemother. 42:2961-2965). Thedolastatin or auristatin drug moiety may be attached to the antibodythrough the N (amino) terminus or the C (carboxyl) terminus of thepeptidic drug moiety (WO 02/088172).

Exemplary auristatin embodiments include the N-terminus linkedmonomethylauristatin drug moieties DE and DF, disclosed in “Senter etal, Proceedings of the American Association for Cancer Research, Volume45, Abstract Number 623, presented Mar. 28, 2004 and described in UnitedStates Patent Publication No. 2005/0238648, the disclosure of which isexpressly incorporated by reference in its entirety.

An exemplary auristatin embodiment is MMAE (see U.S. Pat. No. 6,884,869expressly incorporated by reference in its entirety).

Another exemplary auristatin embodiment is MMAF (see US 2005/0238649,5,767,237 and 6,124,431, expressly incorporated by reference in theirentirety).

Additional exemplary embodiments comprising MMAE or MMAF and variouslinker components (described further herein) have the followingstructures and abbreviations (wherein Ab means antibody and p is 1 toabout 8):

Typically, peptide-based drug moieties can be prepared by forming apeptide bond between two or more amino acids and/or peptide fragments.Such peptide bonds can be prepared, for example, according to the liquidphase synthesis method (see E. Schroder and K. Lubke, “The Peptides”,volume 1, pp 76-136, 1965, Academic Press) that is well known in thefield of peptide chemistry. The auristatin/dolastatin drug moieties maybe prepared according to the methods of: U.S. Pat. No. 5,635,483; U.S.Pat. No. 5,780,588; Pettit et al (1989) J. Am. Chem. Soc. 111:5463-5465;Pettit et al (1998) Anti-Cancer Drug Design 13:243-277; Pettit, G. R.,et al. Synthesis, 1996, 719-725; Pettit et al (1996) J. Chem. Soc.Perkin Trans. 1 5:859-863; and Doronina (2003) Nat Biotechnol21(7):778-784.

Calicheamicin

In other embodiments, the ADC comprises an antibody of the inventionconjugated to one or more calicheamicin molecules. For example, Mylotargis the first commercial ADC drug and utilizes calicheamicin γ1 as thepayload (see U.S. Pat. No. 4,970,198, incorporated by reference in itsentirety). Additional calicheamicin derivatives are described in U.S.Pat. Nos. 5,264,586, 5,384,412, 5,550,246, 5,739,116, 5,773,001,5,767,285 and 5,877,296, all expressly incorporated by reference. Thecalicheamicin family of antibiotics are capable of producingdouble-stranded DNA breaks at sub-picomolar concentrations. For thepreparation of conjugates of the calicheamicin family, see U.S. Pat.Nos. 5,712,374, 5,714,586, 5,739,116, 5,767,285, 5,770,701, 5,770,710,5,773,001, 5,877,296 (all to American Cyanamid Company). Structuralanalogues of calicheamicin which may be used include, but are notlimited to, γ1I, α2I, α2I, N-acetyl-γ1I, PSAG and θI1 (Hinman et al.,Cancer Research 53:3336-3342 (1993), Lode et al., Cancer Research58:2925-2928 (1998) and the aforementioned U.S. patents to AmericanCyanamid). Another anti-tumor drug that the antibody can be conjugatedis QFA which is an antifolate. Both calicheamicin and QFA haveintracellular sites of action and do not readily cross the plasmamembrane. Therefore, cellular uptake of these agents through antibodymediated internalization greatly enhances their cytotoxic effects.

Duocarmycins

CC-1065 (see 4,169,888, incorporated by reference) and duocarmycins aremembers of a family of antitumor antibiotics utilized in ADCs. Theseantibiotics appear to work through sequence-selectively alkylating DNAat the N3 of adenine in the minor groove, which initiates a cascade ofevents that result in apoptosis.

Important members of the duocarmycins include duocarmycin A (U.S. Pat.No. 4,923,990, incorporated by reference) and duocarmycin SA (U.S. Pat.No. 5,101,038, incorporated by reference), and a large number ofanalogues as described in U.S. Pat. Nos. 7,517,903, 7,691,962,5,101,038; 5,641,780; 5,187,186; 5,070,092; 5,070,092; 5,641,780;5,101,038; 5,084,468, 5,475,092, 5,585,499, 5,846,545, WO2007/089149,WO2009/017394A1, 5,703,080, 6,989,452, 7,087,600, 7,129,261, 7,498,302,and 7,507,420, all of which are expressly incorporated by reference.

Other Cytotoxic Agents

Other antitumor agents that can be conjugated to the antibodies of theinvention include BCNU, streptozoicin, vincristine and 5-fluorouracil,the family of agents known collectively LL-E33288 complex described inU.S. Pat. Nos. 5,053,394, 5,770,710, as well as esperamicins (U.S. Pat.No. 5,877,296).

Enzymatically active toxins and fragments thereof which can be usedinclude diphtheria A chain, nonbinding active fragments of diphtheriatoxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain,abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordiiproteins, dianthin proteins, Phytolaca americana proteins (PAPI, PAPII,and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonariaofficinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin,enomycin and the tricothecenes. See, for example, WO 93/21232 publishedOct. 28, 1993.

The present invention further contemplates an ADC formed between anantibody and a compound with nucleolytic activity (e.g., a ribonucleaseor a DNA endonuclease such as a deoxyribonuclease; DNase).

For selective destruction of the tumor, the antibody may comprise ahighly radioactive atom. A variety of radioactive isotopes are availablefor the production of radioconjugated antibodies. Examples includeAt211, I131, I125, Y90, Re186, Re188, Sm153, Bi212, P32, Pb212 andradioactive isotopes of Lu.

The radio- or other labels may be incorporated in the conjugate in knownways. For example, the peptide may be biosynthesized or may besynthesized by chemical amino acid synthesis using suitable amino acidprecursors involving, for example, fluorine-19 in place of hydrogen.Labels such as Tc99m or I123, Re186, Re188 and In111 can be attached viaa cysteine residue in the peptide. Yttrium-90 can be attached via alysine residue. The IODOGEN method (Fraker et al (1978) Biochem.Biophys. Res. Commun 80: 49-57 can be used to incorporate Iodine-123.“Monoclonal Antibodies in Immunoscintigraphy” (Chatal, CRC Press 1989)describes other methods in detail.

For compositions comprising a plurality of antibodies, the drug loadingis represented by p, the average number of drug molecules per Antibody.Drug loading may range from 1 to 20 drugs (D) per Antibody. The averagenumber of drugs per antibody in preparation of conjugation reactions maybe characterized by conventional means such as mass spectroscopy, ELISAassay, and HPLC. The quantitative distribution ofAntibody-Drug-Conjugates in terms of p may also be determined.

In some instances, separation, purification, and characterization ofhomogeneous Antibody-Drug-conjugates where p is a certain value fromAntibody-Drug-Conjugates with other drug loadings may be achieved bymeans such as reverse phase HPLC or electrophoresis. In exemplaryembodiments, p is 2, 3, 4, 5, 6, 7, or 8 or a fraction thereof.

The generation of Antibody-drug conjugate compounds can be accomplishedby any technique known to the skilled artisan. Briefly, theAntibody-drug conjugate compounds can include a multispecific antibodyas the Antibody unit, a drug, and optionally a linker that joins thedrug and the binding agent.

A number of different reactions are available for covalent attachment ofdrugs and/or linkers to binding agents. This is can be accomplished byreaction of the amino acid residues of the binding agent, for example,antibody molecule, including the amine groups of lysine, the freecarboxylic acid groups of glutamic and aspartic acid, the sulfhydrylgroups of cysteine and the various moieties of the aromatic amino acids.A commonly used nonspecific methods of covalent attachment is thecarbodiimide reaction to link a carboxy (or amino) group of a compoundto amino (or carboxy) groups of the antibody. Additionally, bifunctionalagents such as dialdehydes or imidoesters have been used to link theamino group of a compound to amino groups of an antibody molecule.

Also available for attachment of drugs to binding agents is the Schiffbase reaction. This method involves the periodate oxidation of a drugthat contains glycol or hydroxy groups, thus forming an aldehyde whichis then reacted with the binding agent. Attachment occurs via formationof a Schiff base with amino groups of the binding agent. Isothiocyanatescan also be used as coupling agents for covalently attaching drugs tobinding agents. Other techniques are known to the skilled artisan andwithin the scope of the present invention.

In some embodiments, an intermediate, which is the precursor of thelinker, is reacted with the drug under appropriate conditions. In otherembodiments, reactive groups are used on the drug and/or theintermediate. The product of the reaction between the drug and theintermediate, or the derivatized drug, is subsequently reacted with anmultispecific antibody of the invention under appropriate conditions.

It will be understood that chemical modifications may also be made tothe desired compound in order to make reactions of that compound moreconvenient for purposes of preparing conjugates of the invention. Forexample a functional group e g amine, hydroxyl, or sulfhydryl, may beappended to the drug at a position which has minimal or an acceptableeffect on the activity or other properties of the drug

Linker Units

Typically, the antibody-drug conjugate compounds comprise a Linker unitbetween the drug unit and the antibody unit. In some embodiments, thelinker is cleavable under intracellular or extracellular conditions,such that cleavage of the linker releases the drug unit from theantibody in the appropriate environment. For example, solid tumors thatsecrete certain proteases may serve as the target of the cleavablelinker; in other embodiments, it is the intracellular proteases that areutilized. In yet other embodiments, the linker unit is not cleavable andthe drug is released, for example, by antibody degradation in lysosomes.

In some embodiments, the linker is cleavable by a cleaving agent that ispresent in the intracellular environment (for example, within a lysosomeor endosome or caveolea). The linker can be, for example, a peptidyllinker that is cleaved by an intracellular peptidase or protease enzyme,including, but not limited to, a lysosomal or endosomal protease. Insome embodiments, the peptidyl linker is at least two amino acids longor at least three amino acids long or more.

Cleaving agents can include, without limitation, cathepsins B and D andplasmin, all of which are known to hydrolyze dipeptide drug derivativesresulting in the release of active drug inside target cells (see, e.g.,Dubowchik and Walker, 1999, Pharm. Therapeutics 83:67-123). Peptidyllinkers that are cleavable by enzymes that are present inCD38-expressing cells. For example, a peptidyl linker that is cleavableby the thiol-dependent protease cathepsin-B, which is highly expressedin cancerous tissue, can be used (e.g., a Phe-Leu or a Gly-Phe-Leu-Glylinker (SEQ ID NO: 144)). Other examples of such linkers are described,e.g., in U.S. Pat. No. 6,214,345, incorporated herein by reference inits entirety and for all purposes.

In some embodiments, the peptidyl linker cleavable by an intracellularprotease is a Val-Cit linker or a Phe-Lys linker (see, e.g., U.S. Pat.No. 6,214,345, which describes the synthesis of doxorubicin with theval-cit linker).

In other embodiments, the cleavable linker is pH-sensitive, that is,sensitive to hydrolysis at certain pH values. Typically, thepH-sensitive linker hydrolyzable under acidic conditions. For example,an acid-labile linker that is hydrolyzable in the lysosome (for example,a hydrazone, semicarbazone, thiosemicarbazone, cis-aconitic amide,orthoester, acetal, ketal, or the like) may be used. (See, e.g., U.S.Pat. Nos. 5,122,368; 5,824,805; 5,622,929; Dubowchik and Walker, 1999,Pharm. Therapeutics 83:67-123; Neville et al., 1989, Biol. Chem.264:14653-14661.) Such linkers are relatively stable under neutral pHconditions, such as those in the blood, but are unstable at below pH 5.5or 5.0, the approximate pH of the lysosome. In certain embodiments, thehydrolyzable linker is a thioether linker (such as, e.g., a thioetherattached to the therapeutic agent via an acylhydrazone bond (see, e.g.,U.S. Pat. No. 5,622,929).

In yet other embodiments, the linker is cleavable under reducingconditions (for example, a disulfide linker). A variety of disulfidelinkers are known in the art, including, for example, those that can beformed using SATA (N-succinimidyl-5-acetylthioacetate), SPDP(N-succinimidyl-3-(2-pyridyldithio)propionate), SPDB(N-succinimidyl-3-(2-pyridyldithio)butyrate) and SMPT(N-succinimidyl-oxycarbonyl-alpha-methyl-alpha-(2-pyridyl-dithio)toluene)-,SPDB and SMPT. (See, e.g., Thorpe et al., 1987, Cancer Res.47:5924-5931; Wawrzynczak et al., In Immunoconjugates: AntibodyConjugates in Radioimagery and Therapy of Cancer (C. W. Vogel ed.,Oxford U. Press, 1987. See also U.S. Pat. No. 4,880,935.)

In other embodiments, the linker is a malonate linker (Johnson et al.,1995, Anticancer Res. 15:1387-93), a maleimidobenzoyl linker (Lau etal., 1995, Bioorg-Med-Chem. 3(10):1299-1304), or a 3′-N-amide analog(Lau et al., 1995, Bioorg-Med-Chem. 3(10):1305-12).

In yet other embodiments, the linker unit is not cleavable and the drugis released by antibody degradation. (See U.S. Publication No.2005/0238649 incorporated by reference herein in its entirety and forall purposes).

In many embodiments, the linker is self-immolative. As used herein, theterm “self-immolative Spacer” refers to a bifunctional chemical moietythat is capable of covalently linking together two spaced chemicalmoieties into a stable tripartite molecule. It will spontaneouslyseparate from the second chemical moiety if its bond to the first moietyis cleaved. See for example, WO 2007059404A2, WO06110476A2,WO05112919A2, WO2010/062171, WO09/017394, WO07/089149, WO 07/018431,WO04/043493 and WO02/083180, which are directed to drug-cleavablesubstrate conjugates where the drug and cleavable substrate areoptionally linked through a self-immolative linker and which are allexpressly incorporated by reference.

Often the linker is not substantially sensitive to the extracellularenvironment. As used herein, “not substantially sensitive to theextracellular environment,” in the context of a linker, means that nomore than about 20%, 15%, 10%, 5%, 3%, or no more than about 1% of thelinkers, in a sample of antibody-drug conjugate compound, are cleavedwhen the antibody-drug conjugate compound presents in an extracellularenvironment (for example, in plasma).

Whether a linker is not substantially sensitive to the extracellularenvironment can be determined, for example, by incubating with plasmathe antibody-drug conjugate compound for a predetermined time period(for example, 2, 4, 8, 16, or 24 hours) and then quantitating the amountof free drug present in the plasma.

In other, non-mutually exclusive embodiments, the linker promotescellular internalization. In certain embodiments, the linker promotescellular internalization when conjugated to the therapeutic agent (thatis, in the milieu of the linker-therapeutic agent moiety of theantibody-drug conjugate compound as described herein). In yet otherembodiments, the linker promotes cellular internalization whenconjugated to both the auristatin compound and the multispecificantibodies of the invention.

A variety of exemplary linkers that can be used with the presentcompositions and methods are described in WO 2004-010957, U.S.Publication No. 2006/0074008, U.S. Publication No. 20050238649, and U.S.Publication No. 2006/0024317 (each of which is incorporated by referenceherein in its entirety and for all purposes).

Drug Loading

Drug loading is represented by p and is the average number of Drugmoieties per antibody in a molecule. Drug loading (“p”) may be 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or moremoieties (D) per antibody, although frequently the average number is afraction or a decimal. Generally, drug loading of from 1 to 4 isfrequently useful, and from 1 to 2 is also useful. ADCs of the inventioninclude collections of antibodies conjugated with a range of drugmoieties, from 1 to 20. The average number of drug moieties per antibodyin preparations of ADC from conjugation reactions may be characterizedby conventional means such as mass spectroscopy and, ELISA assay.

The quantitative distribution of ADC in terms of p may also bedetermined. In some instances, separation, purification, andcharacterization of homogeneous ADC where p is a certain value from ADCwith other drug loadings may be achieved by means such aselectrophoresis.

For some antibody-drug conjugates, p may be limited by the number ofattachment sites on the antibody. For example, where the attachment is acysteine thiol, as in the exemplary embodiments above, an antibody mayhave only one or several cysteine thiol groups, or may have only one orseveral sufficiently reactive thiol groups through which a linker may beattached. In certain embodiments, higher drug loading, e.g. p>5, maycause aggregation, insolubility, toxicity, or loss of cellularpermeability of certain antibody-drug conjugates. In certainembodiments, the drug loading for an ADC of the invention ranges from 1to about 8; from about 2 to about 6; from about 3 to about 5; from about3 to about 4; from about 3.1 to about 3.9; from about 3.2 to about 3.8;from about 3.2 to about 3.7; from about 3.2 to about 3.6; from about 3.3to about 3.8; or from about 3.3 to about 3.7. Indeed, it has been shownthat for certain ADCs, the optimal ratio of drug moieties per antibodymay be less than 8, and may be about 2 to about 5. See US 2005-0238649A1 (herein incorporated by reference in its entirety).

In certain embodiments, fewer than the theoretical maximum of drugmoieties are conjugated to an antibody during a conjugation reaction. Anantibody may contain, for example, lysine residues that do not reactwith the drug-linker intermediate or linker reagent, as discussed below.Generally, antibodies do not contain many free and reactive cysteinethiol groups which may be linked to a drug moiety; indeed most cysteinethiol residues in antibodies exist as disulfide bridges. In certainembodiments, an antibody may be reduced with a reducing agent such asdithiothreitol (DTT) or tricarbonylethylphosphine (TCEP), under partialor total reducing conditions, to generate reactive cysteine thiolgroups. In certain embodiments, an antibody is subjected to denaturingconditions to reveal reactive nucleophilic groups such as lysine orcysteine.

The loading (drug/antibody ratio) of an ADC may be controlled indifferent ways, e.g., by: (i) limiting the molar excess of drug-linkerintermediate or linker reagent relative to antibody, (ii) limiting theconjugation reaction time or temperature, (iii) partial or limitingreductive conditions for cysteine thiol modification, (iv) engineeringby recombinant techniques the amino acid sequence of the antibody suchthat the number and position of cysteine residues is modified forcontrol of the number and/or position of linker-drug attachments (suchas thioMab or thioFab prepared as disclosed herein and in WO2006/034488(herein incorporated by reference in its entirety)).

It is to be understood that where more than one nucleophilic groupreacts with a drug-linker intermediate or linker reagent followed bydrug moiety reagent, then the resulting product is a mixture of ADCcompounds with a distribution of one or more drug moieties attached toan antibody. The average number of drugs per antibody may be calculatedfrom the mixture by a dual ELISA antibody assay, which is specific forantibody and specific for the drug. Individual ADC molecules may beidentified in the mixture by mass spectroscopy and separated by HPLC,e.g. hydrophobic interaction chromatography.

In some embodiments, a homogeneous ADC with a single loading value maybe isolated from the conjugation mixture by electrophoresis orchromatography.

Methods of Determining Cytotoxic Effect of ADCs

Methods of determining whether a Drug or Antibody-Drug conjugate exertsa cytostatic and/or cytotoxic effect on a cell are known. Generally, thecytotoxic or cytostatic activity of an Antibody Drug conjugate can bemeasured by: exposing mammalian cells expressing a target protein of theAntibody Drug conjugate in a cell culture medium; culturing the cellsfor a period from about 6 hours to about 5 days; and measuring cellviability. Cell-based in vitro assays can be used to measure viability(proliferation), cytotoxicity, and induction of apoptosis (caspaseactivation) of the Antibody Drug conjugate.

For determining whether an Antibody Drug conjugate exerts a cytostaticeffect, a thymidine incorporation assay may be used. For example, cancercells expressing a target antigen at a density of 5,000 cells/well of a96-well plated can be cultured for a 72-hour period and exposed to 0.5μCi of 3H-thymidine during the final 8 hours of the 72-hour period. Theincorporation of 3H-thymidine into cells of the culture is measured inthe presence and absence of the Antibody Drug conjugate.

For determining cytotoxicity, necrosis or apoptosis (programmed celldeath) can be measured. Necrosis is typically accompanied by increasedpermeability of the plasma membrane; swelling of the cell, and ruptureof the plasma membrane. Apoptosis is typically characterized by membraneblebbing, condensation of cytoplasm, and the activation of endogenousendonucleases. Determination of any of these effects on cancer cellsindicates that an Antibody Drug conjugate is useful in the treatment ofcancers.

Cell viability can be measured by determining in a cell the uptake of adye such as neutral red, trypan blue, or ALAMAR™ blue (see, e.g., Pageet al., 1993, Intl. J. Oncology 3:473-476). In such an assay, the cellsare incubated in media containing the dye, the cells are washed, and theremaining dye, reflecting cellular uptake of the dye, is measuredspectrophotometrically. The protein-binding dye sulforhodamine B (SRB)can also be used to measure cytoxicity (Skehan et al., 1990, J. Natl.Cancer Inst. 82:1107-12).

Alternatively, a tetrazolium salt, such as MTT, is used in aquantitative colorimetric assay for mammalian cell survival andproliferation by detecting living, but not dead, cells (see, e.g.,Mosmann, 1983, J. Immunol. Methods 65:55-63).

Apoptosis can be quantitated by measuring, for example, DNAfragmentation. Commercial photometric methods for the quantitative invitro determination of DNA fragmentation are available. Examples of suchassays, including TUNEL (which detects incorporation of labelednucleotides in fragmented DNA) and ELISA-based assays, are described inBiochemica, 1999, no. 2, pp. 34-37 (Roche Molecular Biochemicals).

Apoptosis can also be determined by measuring morphological changes in acell. For example, as with necrosis, loss of plasma membrane integritycan be determined by measuring uptake of certain dyes (e.g., afluorescent dye such as, for example, acridine orange or ethidiumbromide). A method for measuring apoptotic cell number has beendescribed by Duke and Cohen, Current Protocols in Immunology (Coligan etal. eds., 1992, pp. 3.17.1-3.17.16). Cells also can be labeled with aDNA dye (e.g., acridine orange, ethidium bromide, or propidium iodide)and the cells observed for chromatin condensation and margination alongthe inner nuclear membrane. Other morphological changes that can bemeasured to determine apoptosis include, e.g., cytoplasmic condensation,increased membrane blebbing, and cellular shrinkage.

The presence of apoptotic cells can be measured in both the attached and“floating” compartments of the cultures. For example, both compartmentscan be collected by removing the supernatant, trypsinizing the attachedcells, combining the preparations following a centrifugation wash step(e.g., 10 minutes at 2000 rpm), and detecting apoptosis (e.g., bymeasuring DNA fragmentation). (See, e.g., Piazza et al., 1995, CancerResearch 55:3110-16).

In vivo, the effect of a therapeutic composition of the multispecificantibody of the invention can be evaluated in a suitable animal model.For example, xenogenic cancer models can be used, wherein cancerexplants or passaged xenograft tissues are introduced into immunecompromised animals, such as nude or SCID mice (Klein et al., 1997,Nature Medicine 3: 402-408). Efficacy can be measured using assays thatmeasure inhibition of tumor formation, tumor regression or metastasis,and the like.

Therapeutic Uses of Heterodimers

The multispecific proteins, particularly the multispecific antibodies ofthe present invention find use in a variety of therapeutic uses. Asdiscussed in FIG. 1 of Kontermann, supra, incorporated herein byreference, there are a number of dual targeting strategies for cancer,inflammation, etc.

Pharmaceutical Formulations, Administration and Dosing

The therapeutic compositions used in the practice of the foregoingmethods can be formulated into pharmaceutical compositions comprising acarrier suitable for the desired delivery method. Suitable carriersinclude any material that when combined with the therapeutic compositionretains the anti-tumor function of the therapeutic composition and isgenerally non-reactive with the patient's immune system. Examplesinclude, but are not limited to, any of a number of standardpharmaceutical carriers such as sterile phosphate buffered salinesolutions, bacteriostatic water, and the like (see, generally,Remington's Pharmaceutical Sciences 16th Edition, A. Osal., Ed., 1980).

Antibody Compositions for In Vivo Administration

Formulations of the antibodies used in accordance with the presentinvention are prepared for storage by mixing an antibody having thedesired degree of purity with optional pharmaceutically acceptablecarriers, excipients or stabilizers (Remington's Pharmaceutical Sciences16th edition, Osol, A. Ed. [1980]), in the form of lyophilizedformulations or aqueous solutions. Acceptable carriers, excipients, orstabilizers are nontoxic to recipients at the dosages and concentrationsemployed, and include buffers such as phosphate, citrate, and otherorganic acids; antioxidants including ascorbic acid and methionine;preservatives (such as octadecyldimethylbenzyl ammonium chloride;hexamethonium chloride; benzalkonium chloride, benzethonium chloride;phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol);low molecular weight (less than about 10 residues) polypeptides;proteins, such as serum albumin, gelatin, or immunoglobulins;hydrophilic polymers such as polyvinylpyrrolidone; amino acids such asglycine, glutamine, asparagine, histidine, arginine, or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugarssuch as sucrose, mannitol, trehalose or sorbitol; salt-formingcounter-ions such as sodium; metal complexes (e.g. Zn-proteincomplexes); and/or non-ionic surfactants such as TWEEN™, PLURONICS™ orpolyethylene glycol (PEG).

The formulation herein may also contain more than one active compound asnecessary for the particular indication being treated, preferably thosewith complementary activities that do not adversely affect each other.For example, it may be desirable to provide antibodies with otherspecificities. Alternatively, or in addition, the composition maycomprise a cytotoxic agent, cytokine, growth inhibitory agent and/orsmall molecule antagonist. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

The active ingredients may also be entrapped in microcapsules prepared,for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsules,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

The formulations to be used for in vivo administration should besterile, or nearly so. This is readily accomplished by filtrationthrough sterile filtration membranes.

Sustained-release preparations may be prepared. Suitable examples ofsustained-release preparations include semipermeable matrices of solidhydrophobic polymers containing the antibody, which matrices are in theform of shaped articles, e.g. films, or microcapsules. Examples ofsustained-release matrices include polyesters, hydrogels (for example,poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides(U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and .gammaethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradablelactic acid-glycolic acid copolymers such as the LUPRON DEPOT™(injectable microspheres composed of lactic acid-glycolic acid copolymerand leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid. Whilepolymers such as ethylene-vinyl acetate and lactic acid-glycolic acidenable release of molecules for over 100 days, certain hydrogels releaseproteins for shorter time periods.

When encapsulated antibodies remain in the body for a long time, theymay denature or aggregate as a result of exposure to moisture at 37° C.,resulting in a loss of biological activity and possible changes inimmunogenicity. Rational strategies can be devised for stabilizationdepending on the mechanism involved. For example, if the aggregationmechanism is discovered to be intermolecular S—S bond formation throughthio-disulfide interchange, stabilization may be achieved by modifyingsulfhydryl residues, lyophilizing from acidic solutions, controllingmoisture content, using appropriate additives, and developing specificpolymer matrix compositions.

Administrative Modalities

The antibodies and chemotherapeutic agents of the invention areadministered to a subject, in accord with known methods, such asintravenous administration as a bolus or by continuous infusion over aperiod of time, by intramuscular, intraperitoneal, intracerobrospinal,subcutaneous, intra-articular, intrasynovial, intrathecal, oral,topical, or inhalation routes. Intravenous or subcutaneousadministration of the antibody is preferred.

Treatment Modalities

In the methods of the invention, therapy is used to provide a positivetherapeutic response with respect to a disease or condition. By“positive therapeutic response” is intended an improvement in thedisease or condition, and/or an improvement in the symptoms associatedwith the disease or condition. For example, a positive therapeuticresponse would refer to one or more of the following improvements in thedisease: (1) a reduction in the number of neoplastic cells; (2) anincrease in neoplastic cell death; (3) inhibition of neoplastic cellsurvival; (5) inhibition (i.e., slowing to some extent, preferablyhalting) of tumor growth; (6) an increased patient survival rate; and(7) some relief from one or more symptoms associated with the disease orcondition.

Positive therapeutic responses in any given disease or condition can bedetermined by standardized response criteria specific to that disease orcondition. Tumor response can be assessed for changes in tumormorphology (i.e., overall tumor burden, tumor size, and the like) usingscreening techniques such as magnetic resonance imaging (MRI) scan,x-radiographic imaging, computed tomographic (CT) scan, bone scanimaging, endoscopy, and tumor biopsy sampling including bone marrowaspiration (BMA) and counting of tumor cells in the circulation.

In addition to these positive therapeutic responses, the subjectundergoing therapy may experience the beneficial effect of animprovement in the symptoms associated with the disease.

Thus for B cell tumors, the subject may experience a decrease in theso-called B symptoms, i.e., night sweats, fever, weight loss, and/orurticaria. For pre-malignant conditions, therapy with an multispecifictherapeutic agent may block and/or prolong the time before developmentof a related malignant condition, for example, development of multiplemyeloma in subjects suffering from monoclonal gammopathy of undeterminedsignificance (MGUS).

An improvement in the disease may be characterized as a completeresponse. By “complete response” is intended an absence of clinicallydetectable disease with normalization of any previously abnormalradiographic studies, bone marrow, and cerebrospinal fluid (CSF) orabnormal monoclonal protein in the case of myeloma.

Such a response may persist for at least 4 to 8 weeks, or sometimes 6 to8 weeks, following treatment according to the methods of the invention.Alternatively, an improvement in the disease may be categorized as beinga partial response. By “partial response” is intended at least about a50% decrease in all measurable tumor burden (i.e., the number ofmalignant cells present in the subject, or the measured bulk of tumormasses or the quantity of abnormal monoclonal protein) in the absence ofnew lesions, which may persist for 4 to 8 weeks, or 6 to 8 weeks.

Treatment according to the present invention includes a “therapeuticallyeffective amount” of the medicaments used. A “therapeutically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve a desired therapeutic result.

A therapeutically effective amount may vary according to factors such asthe disease state, age, sex, and weight of the individual, and theability of the medicaments to elicit a desired response in theindividual. A therapeutically effective amount is also one in which anytoxic or detrimental effects of the antibody or antibody portion areoutweighed by the therapeutically beneficial effects.

A “therapeutically effective amount” for tumor therapy may also bemeasured by its ability to stabilize the progression of disease. Theability of a compound to inhibit cancer may be evaluated in an animalmodel system predictive of efficacy in human tumors.

Alternatively, this property of a composition may be evaluated byexamining the ability of the compound to inhibit cell growth or toinduce apoptosis by in vitro assays known to the skilled practitioner. Atherapeutically effective amount of a therapeutic compound may decreasetumor size, or otherwise ameliorate symptoms in a subject. One ofordinary skill in the art would be able to determine such amounts basedon such factors as the subject's size, the severity of the subject'ssymptoms, and the particular composition or route of administrationselected.

Dosage regimens are adjusted to provide the optimum desired response(e.g., a therapeutic response). For example, a single bolus may beadministered, several divided doses may be administered over time or thedose may be proportionally reduced or increased as indicated by theexigencies of the therapeutic situation. Parenteral compositions may beformulated in dosage unit form for ease of administration and uniformityof dosage. Dosage unit form as used herein refers to physically discreteunits suited as unitary dosages for the subjects to be treated; eachunit contains a predetermined quantity of active compound calculated toproduce the desired therapeutic effect in association with the requiredpharmaceutical carrier.

The specification for the dosage unit forms of the present invention aredictated by and directly dependent on (a) the unique characteristics ofthe active compound and the particular therapeutic effect to beachieved, and (b) the limitations inherent in the art of compoundingsuch an active compound for the treatment of sensitivity in individuals.

The efficient dosages and the dosage regimens for the multispecificantibodies used in the present invention depend on the disease orcondition to be treated and may be determined by the persons skilled inthe art.

An exemplary, non-limiting range for a therapeutically effective amountof an multispecific antibody used in the present invention is about0.1-100 mg/kg, such as about 0.1-50 mg/kg, for example about 0.1-20mg/kg, such as about 0.1-10 mg/kg, for instance about 0.5, about such as0.3, about 1, or about 3 mg/kg. In another embodiment, the antibody isadministered in a dose of 1 mg/kg or more, such as a dose of from 1 to20 mg/kg, e.g. a dose of from 5 to 20 mg/kg, e.g. a dose of 8 mg/kg.

A medical professional having ordinary skill in the art may readilydetermine and prescribe the effective amount of the pharmaceuticalcomposition required. For example, a physician or a veterinarian couldstart doses of the medicament employed in the pharmaceutical compositionat levels lower than that required in order to achieve the desiredtherapeutic effect and gradually increase the dosage until the desiredeffect is achieved.

In one embodiment, the multispecific antibody is administered byinfusion in a weekly dosage of from 10 to 500 mg/kg such as of from 200to 400 mg/kg Such administration may be repeated, e.g., 1 to 8 times,such as 3 to 5 times. The administration may be performed by continuousinfusion over a period of from 2 to 24 hours, such as of from 2 to 12hours.

In one embodiment, the multispecific antibody is administered by slowcontinuous infusion over a long period, such as more than 24 hours, ifrequired to reduce side effects including toxicity.

In one embodiment the multispecific antibody is administered in a weeklydosage of from 250 mg to 2000 mg, such as for example 300 mg, 500 mg,700 mg, 1000 mg, 1500 mg or 2000 mg, for up to 8 times, such as from 4to 6 times. The administration may be performed by continuous infusionover a period of from 2 to 24 hours, such as of from 2 to 12 hours. Suchregimen may be repeated one or more times as necessary, for example,after 6 months or 12 months. The dosage may be determined or adjusted bymeasuring the amount of compound of the present invention in the bloodupon administration by for instance taking out a biological sample andusing anti-idiotypic antibodies which target the antigen binding regionof the multispecific antibody.

In a further embodiment, the multispecific antibody is administered onceweekly for 2 to 12 weeks, such as for 3 to 10 weeks, such as for 4 to 8weeks.

In one embodiment, the multispecific antibody is administered bymaintenance therapy, such as, e.g., once a week for a period of 6 monthsor more.

In one embodiment, the multispecific antibody is administered by aregimen including one infusion of an multispecific antibody followed byan infusion of an multispecific antibody conjugated to a radioisotope.The regimen may be repeated, e.g., 7 to 9 days later.

As non-limiting examples, treatment according to the present inventionmay be provided as a daily dosage of an antibody in an amount of about0.1-100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9,10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on atleast one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 afterinitiation of treatment, or any combination thereof, using single ordivided doses of every 24, 12, 8, 6, 4, or 2 hours, or any combinationthereof.

In some embodiments the multispecific antibody molecule thereof is usedin combination with one or more additional therapeutic agents, e.g. achemotherapeutic agent. Non-limiting examples of DNA damagingchemotherapeutic agents include topoisomerase I inhibitors (e.g.,irinotecan, topotecan, camptothecin and analogs or metabolites thereof,and doxorubicin); topoisomerase II inhibitors (e.g., etoposide,teniposide, and daunorubicin); alkylating agents (e.g., melphalan,chlorambucil, busulfan, thiotepa, ifosfamide, carmustine, lomustine,semustine, streptozocin, decarbazine, methotrexate, mitomycin C, andcyclophosphamide); DNA intercalators (e.g., cisplatin, oxaliplatin, andcarboplatin); DNA intercalators and free radical generators such asbleomycin; and nucleoside mimetics (e.g., 5-fluorouracil, capecitibine,gemcitabine, fludarabine, cytarabine, mercaptopurine, thioguanine,pentostatin, and hydroxyurea).

Chemotherapeutic agents that disrupt cell replication include:paclitaxel, docetaxel, and related analogs; vincristine, vinblastin, andrelated analogs; thalidomide, lenalidomide, and related analogs (e.g.,CC-5013 and CC-4047); protein tyrosine kinase inhibitors (e.g., imatinibmesylate and gefitinib); proteasome inhibitors (e.g., bortezomib); NF-κBinhibitors, including inhibitors of IκB kinase; antibodies which bind toproteins overexpressed in cancers and thereby downregulate cellreplication (e.g., trastuzumab, rituximab, cetuximab, and bevacizumab);and other inhibitors of proteins or enzymes known to be upregulated,over-expressed or activated in cancers, the inhibition of whichdownregulates cell replication.

In some embodiments, the antibodies of the invention can be used priorto, concurrent with, or after treatment with Velcade® (bortezomib).

All cited references are herein expressly incorporated by reference intheir entirety.

Whereas particular embodiments of the invention have been describedabove for purposes of illustration, it will be appreciated by thoseskilled in the art that numerous variations of the details may be madewithout departing from the invention as described in the appendedclaims.

EXAMPLES

Examples are provided below to illustrate the present invention. Theseexamples are not meant to constrain the present invention to anyparticular application or theory of operation. For all constant regionpositions discussed in the present invention, numbering is according tothe EU index as in Kabat (Kabat et al., 1991, Sequences of Proteins ofImmunological Interest, 5th Ed., United States Public Health Service,National Institutes of Health, Bethesda, entirely incorporated byreference). Those skilled in the art of antibodies will appreciate thatthis convention consists of nonsequential numbering in specific regionsof an immunoglobulin sequence, enabling a normalized reference toconserved positions in immunoglobulin families. Accordingly, thepositions of any given immunoglobulin as defined by the EU index willnot necessarily correspond to its sequential sequence.

Example 1 Design of Non-Native Charge Substitutions to Reduce pI

Antibody constant chains were modified with lower pI by engineeringsubstitutions in the constant domains. Reduced pI can be engineered bymaking substitutions of basic amino acids (K or R) to acidic amino acids(D or E), which result in the largest decrease in pI. Mutations of basicamino acids to neutral amino acids and neutral amino acids to acidicamino acids will also result in a decrease in pI. A list of amino acidpK values can be found in Table 1 of Bjellqvist et al., 1994,Electrophoresis 15:529-539.

We chose to explore substitutions in the antibody CH1 (Cγ1) and CL(Ckappa or CK) regions (sequences are shown in FIG. 1) because, unlikethe Fc region, they do not interact with native ligands that impact theantibody's pharmacological properties. In deciding which positions tomutate, the surrounding environment and number of contacts the WT aminoacid makes with its neighbors was taken into account such as to minimizethe impact of a substitution or set of substitutions on structure and/orfunction. The solvent accessibility or fraction exposed of each CH1 andCK position was calculated using relevant crystal structures of antibodyFab domains. The results are shown in FIGS. 2 and 3 for the Cγ1 and CKrespectively. Design was guided further by examining the CH1 and CLdomains for positions that are isotypic between the immunoglobulinisotypes (IgG1, IgG2, IgG3, and IgG4). Because such variations occurnaturally, such position are expected to be amenable to substitution.Based on this analysis, a number of substitutions were identified thatreduce pI but are predicted to have minimal impact on the biophysicalproperties of the domains.

Example 2 Anti-VEGF Antibodies with Engineered CH1 and CK Regions HavingLower pI

Amino acid modifications were engineered in the CH1 and CK domains of anIgG1 antibody to lower the pI of the antibody. Based on the aboveanalysis, chosen substitutions for the heavy chain CH1 were 119E, 133E,164E, 205E, 208D, and 210E, and substitutions for the light chain CKsubstitutions were 126E, 145E, 152D, 156E, 169E, and 202E. These variantconstant chains are referred to as IgG1-CH1-pI(6) and CK-pI(6)respectively, and their amino acid sequences are provided in FIG. 4.

CH1 and CK variants were engineered in the context of an antibodytargeting vascular endothelial factor (VEGF). The heavy and light chainvariable regions (VH and VL) are those of a humanized version of theantibody A4.6.1, also referred to as bevacizumab (Avastin®), which isapproved for the treatment of a variety of cancers. These variableregion sequences are provided in FIG. 5. The anti-VEGF antibody variantcontaining the low pI substitutions is referred to as XENP9493Bevacizumab-IgG1-CH1-pI(6)-CK-pI(6), and the amino acid sequences of theheavy and light chains of this antibody are provided in FIG. 6. Astructural model of the Fab domain showing the 6 substitutions ofCH1-pI(6) and the 6 substitutions of CK-pI(6) is shown in FIG. 7. Thecalculated pI of WT anti-VEGF (bevacizumab) is 8.14. The calculated pIof the engineered anti-VEGF CH1 variant is 6.33 and that of theanti-VEGF CK variant is 6.22. When the heavy chain and light chain pIengineered anti-VEGF variants are co-transfected, the full-lengthanti-VEGF mAb has a calculated pI of 5.51.

Genes encoding the heavy and light chains of the anti-VEGF antibodieswere constructed in the mammalian expression vector pTT5. The human IgG1constant chain gene was obtained from IMAGE clones and subcloned intothe pTT5 vector. VH and VL genes encoding the anti-VEGF antibodies weresynthesized commercially (Blue Heron Biotechnologies, Bothell Wash.),and subcloned into the vectors encoding the appropriate CL and IgG1constant chains. Amino acid modifications were constructed usingsite-directed mutagenesis using the QuikChange® site-directedmutagenesis methods (Stratagene, La Jolla Calif.). All DNA was sequencedto confirm the fidelity of the sequences.

Plasmids containing heavy chain gene (VH-Cγ1-Cγ2-Cγ3) wereco-transfected with plasmid containing light chain gene (VL-CK) into293E cells using lipofectamine (Invitrogen, Carlsbad Calif.) and grownin FreeStyle 293 media (Invitrogen, Carlsbad Calif.). After 5 days ofgrowth, the antibodies were purified from the culture supernatant byprotein A affinity using the MabSelect resin (GE Healthcare). Antibodyconcentrations were determined by bicinchoninic acid (BCA) assay(Pierce).

The pI engineered anti-VEGF mAbs were characterized by SDS PAGE on anAgilent Bioanalyzer (FIG. 8), by size exclusion chromatography (SEC)(FIG. 9), isoelectric focusing (IEF) gel electrophoresis (FIG. 10),binding to antigen VEGF by Biacore (FIG. 11), and differential scanningcalorimetry (DSC) (FIG. 12). All mAbs showed high purity on SDS-PAGE andSEC. IEF gels indicated that each variant had the designed isoelectricpoint. VEGF binding analysis on Biacore showed that pI engineeredanti-VEGF bound to VEGF with similar affinity as bevacizumab, indicatingthat the designed substitutions did not perturb the function of the mAb.DSC showed that the anti-VEGF variant with both CH1 and CL engineeredsubstitutions had high thermostability with a Tm of 71.9° C.

Pharmacokinetic experiments were performed in B6 mice that arehomozygous knock-outs for murine FcRn and heterozygous knock-ins ofhuman FcRn (mFcRn−/−, hFcRn+) (Petkova et al., 2006, Int Immunol18(12):1759-69, entirely incorporated by reference), herein referred toas hFcRn or hFcRn+ mice. Samples tested included the parent IgG1/2constant region, the pI-engineered variant with a pI of 5.51, referredto as IgG1_CH-CL_pI_eng, and an Fc variant version of IgG1/2 containingthe substitution N434S, which improves affinity to human FcRn.

A single, intravenous tail vein injection of anti-VEGF antibody (2mg/kg) was given to groups of 4-7 female mice randomized by body weight(20-30 g range). Blood (˜50 ul) was drawn from the orbital plexus ateach time point, processed to serum, and stored at −80° C. untilanalysis. Antibody concentrations were determined using an ELISA assay.Serum concentration of antibody was measured using a recombinant VEGF(VEGF-165, PeproTech, Rocky Hill, N.J.) as capture reagent, anddetection was carried out with biotinylated anti-human kappa antibodyand europium-labeled streptavidin. The time resolved fluorescence signalwas collected. PK parameters were determined for individual mice with anon-compartmental model using WinNonLin (Pharsight Inc, Mountain ViewCalif.). Nominal times and dose were used with uniform weighing ofpoints.

Results are shown in FIG. 13. Fitted half-life (t½) values, whichrepresents the beta phase that characterizes elimination of antibodyfrom serum, are shown in Table 1. The pI-engineered variant, containingsubstitutions in CH 1 and CL that reduce the pI, extended half-life to7.4 days, an improvement of approximately 2.6-fold relative to IgG1/2.The pI-engineered variant had a comparable half-life to the Fc variantversion N434S. Combinations of antibody variants are contemplated thatreduce pI and improve affinity for FcRn for extending the half-lives ofantibodies and Fc fusions.

TABLE 1 PK results of pI-engineered variant Average St. Individual micet½ (days) t½ Dev. Group Variant n n1 n2 n3 n4 (days) (days) 7349IgG1/2_WT 4 2.9 2.5 3.2 2.8 2.9 0.3 7350 IgG1/2_N434S 4 6.3 7.7 7.3 6.57.0 0.7 9493 IgG1_CH- 3 7.4 8.4 6.4 7.4 1.0 CL_pI_eng

Example 3 PK Analysis of IgG Constant Regions

PK studies of IgG1 and IgG2 isotype versions of bevacizumab were carriedout in the huFcRn mice as described above. The IgG1 results from fourseparate PK studies are shown in FIG. 14. The half-lives from the fourstudies were 3.0, 3.9, 2.8, and 2.9 days, resulting in an averagehalf-life of 3.2 days. The PK results from the IgG2 study are shown inFIG. 15. The half-life of IgG2 was 5.9 days.

The PK results from the IgG1 and IgG2 were analyzed with the resultsfrom the IgG½ and pI-engineered versions of bevacizumab. Table 2 showsthe half-lives of the antibodies along with their calculated pI. Thesedata are plotted in FIG. 16.

TABLE 2 PK results of antibodies with identical Fv (bevacizumab) butconstant regions with different pI's XENP IgG pI Average t½ (days) 4547IgG1 8.1 3.2 7349 IgG1/2 8.1 2.9 6384 IgG2 7.3 5.9 9493IgG1_CH-CL_pI_eng 5.6 7.4 [aka IgG1-pI(12)]

A correlation was observed between half-life and the pI of theantibodies. These data further suggest that engineering of antibodyconstant chains, including heavy and light chain constant regions, forreduced isoelectric point is potentially a novel generalizable approachto extending the serum half-lives of antibodies and Fc fusions.

Example 4 Engineering Approaches to Constant Region pI Engineering

Reduction in the pI of a protein or antibody can be carried out using avariety of approaches. At the most basic level, residues with high pKa's(lysine, arginine, and to some extent histidine) are replaced withneutral or negative residues, and/or neutral residues are replaced withlow pKa residues (aspartic acid and glutamic acid). The particularreplacements may depend on a variety of factors, including location inthe structure, role in function, and immunogenicity.

Because immunogenicity is a concern, efforts can be made to minimize therisk that a substitution that lowers the pI will elicit immunogenicity.One way to minimize risk is to minimize the mutational load of thevariants, i.e. to reduce the pI with the fewest number of mutations.Charge swapping mutations, where a K, R, or H is replaced with a D or E,have the greatest impact on reducing pI, and so these substitutions arepreferred. Another approach to minimizing the risk of immunogenicitywhile reducing pI is to utilize substitutions from homologous humanproteins. Thus for antibody constant chains, the isotypic differencesbetween the IgG subclasses (IgG1, IgG2, IgG3, and IgG4) provide low-risksubstitutions. Because immune recognition occurs at a local sequencelevel, i.e. MHC II and T-cell receptors recognize epitopes typically 9residues in length, pI-altering substitutions may be accompanied byisotypic substitutions proximal in sequence. In this way, epitopes canbe extended to match a natural isotype. Such substitutions would thusmake up epitopes that are present in other human IgG isotypes, and thuswould be expected to be tolerized.

FIG. 17 shows an amino acid sequence alignment of the IgG subclasses.Residues with a bounded box illustrate isotypic differences between theIgG's. Residues which contribute to a higher pI (K, R, and H) or lowerpI (D and E) are highlighted in bold. Designed substitutions that eitherlower the pI, or extend an epitope to match a natural isotype are shownin gray.

FIG. 18 shows the amino acid sequence of the CK and Cλ light constantchains. Homology between Cκ and Cλ is not as high as between the IgGsubclasses. Nonetheless the alignment may be used to guidesubstitutions. Residues which contribute to a higher pI (K, R, and H) orlower pI (D and E) are highlighted in bold. Gray indicates lysine,arginines, and histidines that may be substituted, preferably withaspartic or glutamic acids, to lower the isoelectric point.

Another approach to engineering lower pI into proteins and antibodies isto fuse negatively charged residues to the N- or C-termini. Thus forexample, peptides consisting principally of aspartic acids and glutamicacid may be fused to the N-terminus or C-terminus to the antibody heavychain, light chain or both. Because the N-termini are structurally closeto the antigen binding site, the C-termini are preferred.

Based on the described engineering approaches, a number of variants weredesigned to reduce the isoelectric point of both the antibody heavychain and light chain. The heavy chain variants comprise variouscombinations of isotypic substitutions, as well as C-terminal negativelycharged peptides. Relative to a native IgG1, the variants comprise oneor more isotypic substitutions from the group consisting of G137E,G138S, S192N, L193F, I199T, N203D, K214T, K222T, substitution of 221-225DKTHT to VE, H268Q, K274Q, R355Q, N384S, K392N, V397M, Q419E, and adeletion of K447 (referred to as K447#), wherein numbering is accordingto the EU index. The light chain variants comprise various combinationsof non-isotypic substitutions and C-terminal negatively chargedpeptides. CK variants comprise one or more substitutions from the groupconsisting of K126E, K145E, N152D, S156E, K169E, and S202E, whereinnumbering is according to the EU index.

Sequences of the variant heavy chains are provided in FIG. 19, andsequences of the variant light chains are provided in FIG. 20. Table 3lists the variants constructed, along with the calculated pI's of theheavy constant chain, light constant chain, as well as the pI of thefull length monoclonal antibody (mAb) containing the variable region(Fv) of the anti-VEGF antibody Bevacizumab.

TABLE 3 pI-engineered antibody constant chain variants Fv Heavy ChainLight Chain VH VL mAb^(b) Identity pI Identity pI Identity^(a) pI pI pIIgG1-WT 8.46 Ck-WT 6.1 Bev 6.99 6.75 8.10 IgG1-WT 8.46 Ck-pI(3) 4.6 Bev6.99 6.75 6.58 IgG1-WT 8.46 Ck-pI(6) 4.4 Bev 6.99 6.75 6.21 IgG1-WT 8.46Ck-pI(6- 4.3 Bev 6.99 6.75 5.85 DEDE) IgG2-WT 7.66 Ck-WT 6.1 Bev 6.996.75 7.31 IgG2-WT 7.66 Ck-pI(3) 4.6 Bev 6.99 6.75 6.16 IgG2-WT 7.66Ck-pI(6) 4.4 Bev 6.99 6.75 5.88 IgG2-WT 7.66 Ck-pI(6- 4.3 Bev 6.99 6.755.58 DEDE) pI-iso1 5.93 Ck-WT 6.1 Bev 6.99 6.75 6.16 pI-iso1(NF) 5.93Ck-WT 6.1 Bev 6.99 6.75 6.16 pI-iso1(NF- 5.85 Ck-WT 6.1 Bev 6.99 6.756.11 VE) pI-iso1(NF- 5.85 Ck-pI(3) 4.6 Bev 6.99 6.75 5.58 VE)pI-iso1(NF- 5.85 Ck-pI(6) 4.4 Bev 6.99 6.75 5.38 VE) pI-iso1(NF- 5.85Ck-pI(6- 4.3 Bev 6.99 6.75 5.18 VE) DEDE) pI-iso1(NF- 5.36 Ck-WT 6.1 Bev6.99 6.75 5.74 VE-DEDE) pI-iso1(NF- 5.36 Ck-pI(3) 4.6 Bev 6.99 6.75 5.32VE-DEDE) pI-iso1(NF- 5.36 Ck-pI(6) 4.4 Bev 6.99 6.75 5.18 VE-DEDE)pI-iso1(NF- 5.36 Ck-pI(6- 4.3 Bev 6.99 6.75 5.03 VE-DEDE) DEDE) ^(a)Bev= the variable region of the anti-VEGF antibody Bevacizumab ^(b)mAb pI =the pI of the full length monoclonal antibody containing the Fv ofBevacizumab

Example 5 Determination of Charge-Dependency of pI Engineering andPotential Combination with Fc Variants that Enhance Binding to FcRn

A series of new pI-engineered variants were generated to test twoaspects of the relationship between low pI and extended half-life.First, the parameter of charge was investigated by making a controlledset of variants based on the 9493 IgG1-pI(12) variant. These variants,10017, 10018, and 10019, are described in Table 4, along with their pIand the differences in positively and negatively charged residuesrelative to bevacizumab IgG1 WT.

TABLE 4 Engineered constructs exploring charge and Fc variants HC LCCharge XENP HC Identity Substitutions Substitutions PI State # KR # DE4547 IgG1-WT 8.1  (+6)  0  0 9493 IgG1-pI(12) CH1-pI(6) Ck-pI(6) 5.6(−30) (−12) (+24) 9992 IgG1-pI(12) CH1-pI(6) + Ck-pI(6) 5.6 (−30) (−12)(+24) N434S 9993 IgG1-pI(12) CH1-pI(6) + Ck-pI(6) 5.6 (−30) (−12) (+24)M428L/N434S 10017 IgG1-pI(6)- S119E T164E N152D S156E 6.6  (−6)  0 (+12)Neutral-to-DE N208D S202E 10018 IgG1-pI(6)- K133Q K205Q K126Q K145Q 6.6 (−6) (−12)  0 KR-to-Neutral K210Q K169Q 10019 IgG1-pI(6)- K133E K205EK126E K145E 5.9 (−18) (−12) (+12) KR-to-DE K210E K169E CH1-pI(6) = S119EK133E T164E K205E N208D K210E Ck-pI(6) = K126E K145E N152D S156E K169ES202E pI calculated with Fv = Bevacizumab

The experimental rationale here is as follows. If all the mechanism forimproved half-life is based on removal of positive charge, 10018 and10019 should be as good as 9493 while 10017 would not be extended. Ifthe mechanism is based on an increase in negative charge, 10018 will notbe extended, while 10017 and 10019 will have equivalent half-life thatis extended relative to IgG1 but shorter than 9493. If overall pI (orcharge state) is the basis, the result will be 9493>10019>10017=10018.

In addition to the charge-controlled variant set, the 9493 IgG1-pI(12)variant was combined with substitutions that improve binding to FcRn atpH 6.0 in order to test whether the two mechanisms of half-lifeimprovement, charge state and FcRn, are compatible. These variants, 9992IgG1-p1(12)-N4345 and 9993 IgG1-p1(12)-M428L/N434S, are listed in Table4.

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations are plotted in FIGS. 21 and 22, along with thehalf-lives obtained from the fits of the data.

The results indicate that both reducing positive charge and increasingnegative charge contribute to improved half-life. In addition, theresults indicate that engineered lower pI and increased binding to FcRncan be used in combination to obtain even greater enhancements inhalf-life. A plot of the half-life vs. pI relationship is provided inFIG. 23 for variant and native IgG's of identical Fv (bevacizumab) thathave been tested in the huFcRn mice. The graph illustrates again theinverse relationship between half-life and pI, as well as thecombinability of variants engineered for lower pI and Fc variants thatimprove binding to FcRn.

Example 6 New pI-Engineered Constructs

As described above, efforts can be made to minimize the risk thatsubstitutions that lower pI will elicit immunogenicity by utilizing theisotypic differences between the IgG subclasses (IgG1, IgG2, IgG3, andIgG4). A new set of novel isotypes was designed based on this principal.Again, because immune recognition occurs at a local sequence level, i.e.MHC II and T-cell receptors recognize epitopes typically 9 residues inlength, pI-altering substitutions were accompanied by isotypicsubstitutions proximal in sequence. In this way, epitopes were extendedto match a natural isotype. Such substitutions would thus make upepitopes that are present in other human IgG isotypes, and thus would beexpected to be tolerized.

The designed low-pI isotypes, referred to as IgG-pI-Iso2,IgG-pI-Iso2-SL, IgG-pI-Iso2-charges-only, IgG-pI-Iso3, IgG-pI-Iso3-SL,and IgG-pI-Iso3-charges-only are described in Table 5, along with theirpI and effector function properties. FIG. 24 provides a sequencealignment of IgG-pI-Iso3 with the native IgG isotypes, and depictsresidue identities and residues that reduce pI relative to one or moreof the native IgG isotypes. FIGS. 25 and 26 illustrate the structuraldifferences between IgG1 and IgG-pI-Iso3. IgG-pI-Iso2, IgG-pI-Iso2-SL,and IgG-pI-Iso2-charges-only were designed to have low (weak) effectorfunction, as determined by IgG2-like residues in the hinge (233P, 234V,235A) and CH2 domain (327G). IgG-pI-Iso3, IgG-pI-Iso3-SL, andIgG-pI-Iso3-charges-only were designed to have high (strong) effectorfunction, as determined by IgG1-like residues in the hinge (233E, 234L,235L, 236G) and CH2 domain (327A). Isotypic low pI variants with the“SL” designation indicate that these variants differ from IgG-pI-Iso2and IgG-pI-Iso3 by having 192S and 193L. Serine and leucine at thesepositions were found to be more compatible than 192N/193F due todifferences in neighboring residues that are present in IgG1 and IgG2.Low pI isotype variants designated as “charges only” contain chargeaffecting isotypic substitutions, but do not contain the neighboringnon-charge altering substitutions. The novel isotypes can be combinedwith a native light chain constant region (Ckappa or Clambda), or avariant version engineered with substitutions to further reduce the pI.An example of a pI-engineered light constant chain is a new variantreferred to as CK-pI(4), described schematically in FIG. 27. Inaddition, the novel isotypes can be engineered with Fc variants thatimprove affinity to FcRn, thereby further enabling extended half-life.Such Fc variants may include, for example 434S or 428L/4345 as describedin Table 5, or other Fc variants as described herein Amino acidsequences of IgG-pI-Iso2, IgG-pI-Iso2-SL, IgG-pI-Iso2-charges-only,IgG-pI-Iso3, IgG-pI-Iso3-SL, IgG-pI-Iso3-charges-only and CK-pI(4) areprovided in FIG. 28.

TABLE 5 Novel IgG isotypes with low pI Effector XENP Heavy Light Fcvariant pI Function 10178 IgG-pI-Iso2 WT 6.3 Low 10470 IgG-pI-Iso2-SL WT6.3 Low 10180 IgG-pI-Iso2 WT 434S 6.3 Low 10471 IgG-pI-Iso2-SL WT 434S6.3 Low 10182 IgG-pI-Iso2 CK-pI(4) 5.6 Low 10184 IgG-pI-Iso2 CK-pI(4)434S 5.6 Low 10427 IgG-pI-Iso2-charges- WT 6.3 Low only 10473IgG-pI-Iso2-charges- WT 434S 6.3 Low only 10179 IgG-pI-Iso3 WT 6.2 High10286 IgG-pI-Iso3-SL WT 6.2 High 10181 IgG-pI-Iso3 WT 434S 6.2 High10466 IgG-pI-Iso3-SL WT 434S 6.2 High 10467 IgG-pI-Iso3-SL WT 428L/ 6.2High 434S 10183 IgG-pI-Iso3 CK-pI(4) 5.5 High 10185 IgG-pI-Iso3 CK-pI(4)434S 5.5 High 10525 IgG-pI-Iso3-SL CK-pI(4) 434S 5.5 High 10426IgG-pI-Iso3-charges- WT 6.2 High only 10472 IgG-pI-Iso3-charges- WT 434S6.2 High only SL = 192S/193L CK-pI(4) = K126E/K145E/K169E/K207E pIcalculated with Fv = Bevacizumab

The novel engineered isotypes can be combined with other Fc variants togenerate antibodies or Fc fusions with extended half-life and otherimproved properties. For example, IgG-pI-Iso2-SL and/or IgG-pI-Iso3-SLmay incorporate variants 239D, 332E, 267E, and/or 328F that modulatebinding to FcγRs to provide enhanced effector function orimmunomodulatory properties (as well as other variants listed in LegendB of FIG. 83. The novel isotypes may be combined with other Fc variantsthat improve binding to FcRn, including for example 428L, 428L/434S,T250Q/M428L, M252Y/S254T/T256E, and N434A/T307Q, (and others listed inLegend A of FIG. 83) thereby potentially further extending in vivohalf-life. Exemplary heavy chains are described in Table 6. Suchvariants may be expressed with a light chain that has a native constantlight chain (CK or C4 or one that also incorporates constant light chainmodifications that reduce pI, including for example any of theengineered constant light chains described herein, including for exampleCK-pI(4).

TABLE 6 Engineered combinations of pI isotype variants with othervariants. Heavy Fc IgG-pI-Iso3-SL 332E IgG-pI-Iso3-SL 239D/332EIgG-pI-Iso3-SL 332E/434S IgG-pI-Iso3-SL 239D/332E/434S IgG-pI-Iso2-SL267E/328F IgG-pI-Iso2-SL 434S/267E/328F IgG-pI-Iso3-SL 267E/328FIgG-pI-Iso3-SL 434S/267E/328F IgG-pI-Iso2-SL 428L/434S IgG-pI-Iso3-SL428L/434S IgG-pI-Iso2-SL 428L IgG-pI-Iso3-SL 428L IgG-pI-Iso2-SL250Q/428L IgG-pI-Iso3-SL 250Q/428L IgG-pI-Iso2-SL 252Y/254T/256EIgG-pI-Iso3-SL 252Y/254T/256E IgG-pI-Iso2-SL 434A/307Q IgG-pI-Iso3-SL434A/307Q

In order to reduce pI even further, additional variant heavy constantchains with reduced pI were designed to minimize mutational load byintroducing charge swapping mutations, i.e. where K and R were replacedwith D or E, as described above. To aid in the design of these variants,fraction exposed as well as the energy change upon substitution to Gluwere calculated for each K and R residue in the Fc region (FIG. 29).These new variants are referred to as pI(7) and pI(11). pI(7)incorporated amino acid modifications K133E, K205E, K210E, K274E, R355E,K392E, and a deletion of the Lys at 447, and pI(11) incorporated aminoacid modifications K133E, K205E, K210E, K274E, K320E, K322E, K326E,K334E, R355E, K392E, and a deletion of the Lys at 447 Thesemodifications were introduced into heavy constant chains to result inantibodies with strong effector function, IgG1-pI(7) and IgG1-pI(11),and weak effector function IgG1/2-pI(7) and IgG1/2-pI(11). As can beseen in FIG. 30, as mAb pI gets lower, it requires a greater number ofcharge swap substitutions to decrease pI further. These pI-engineeredvariants are described in Table 7, and amino acid sequences are providedin FIG. 28.

TABLE 7 Engineered charge swaps Fc XENP Heavy variant Light pI 10107IgG1-pI(7) CK-pI(4) 5.3 10108 IgG1-pI(11) CK-pI(4) 5.0 10109IgG1/2-pI(7) CK-pI(4) 5.4 10110 IgG1/2-pI(11) CK-pI(4) 5.0 10476IgG1/2-pI(7) 434S CK-pI(4) 5.4 IgG1-pI(7) =K133E/K205E/K210E/K274E/R355E/K392E/K447# IgG1-pI(11) =K133E/K205E/K210E/K274E/K320E/K322E/K326E/K334E/R355E/K392E/K447#IgG1/2-pI(7) = K133E/K205E/K210E/Q274E/R355E/K392E/K447# IgG1/2-pI(11) =K133E/K205E/K210E/Q274E/K320E/K322E/K326E/K334E/R355E/K392E/K447#CK-pI(4) = K126E/K145E/K169E/K207E pI calculated with Fv = Bevacizumab

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations are plotted in FIG. 31 and FIG. 32, along with thehalf-lives obtained from the fits of the data. Half-lives for individualmice are plotted in FIG. 33. The data clearly demonstrate the additivityof low pI from isotypic pI variants as well as enhanced FcRn bindingfrom the N434S substitution as shown by a plot of half-life vs. pI asshown in FIG. 34.

Example 7 Isotypic Light Chain Constant Region Variants

Homology between CK and C2, is not as high as between the IgG subclasses(as shown in FIG. 18), however the sequence and structural homology thatexists may still be used to guide substitutions to create an isotypiclow-pI light chain constant region. In FIG. 18, positions with residuescontributing to a higher pI (K, R, and H) or lower pI (D and E) arehighlighted in bold. Gray indicates lysine, arginines, and histidinesthat may be substituted, preferably with aspartic or glutamic acids, tolower the isoelectric point. A structural alignment of CK and Cλ, wasconstructed (FIG. 35) and used along with the sequence alignment as aguide to make several CK/C?, isotypic variants. These pI-engineeredvariants are described in Table 8, and amino acid sequences are providedin FIG. 28.

TABLE 8 Engineered low-pI variants containing isotypic light chainconstant regions Effector XENP Heavy Light Fc variant pI Function 10324IgG-pI-Iso3 CK-Iso(3) 5.9 High 10325 IgG-pI-Iso3 CK-Iso(4) 5.8 High10326 IgG-pI-Iso3 CK-Iso(5) 5.8 High 10327 IgG-pI-Iso3 CK-Iso(6) 5.7High 10511 IgG-pI-Iso3-SL CK-Iso(3) 5.9 High 10512 IgG-pI-Iso3-SLCK-Iso(4) 5.8 High 10513 IgG-pI-Iso3-SL CK-Iso(5) 5.8 High 10517IgG-pI-Iso3-SL CK-Iso(3) 434S 5.9 High 10518 IgG-pI-Iso3-SL CK-Iso(4)434S 5.8 High 10519 IgG-pI-Iso3-SL CK-Iso(5) 434S 5.8 High 10520IgG-pI-Iso3-SL CK-Iso(3) 428L/434S 5.9 High 10521 IgG-pI-Iso3-SLCK-Iso(4) 428L/434S 5.8 High 10522 IgG-pI-Iso3-SL CK-Iso(5) 428L/434S5.8 High 10526 IgG-pI-Iso3 CK-Iso(5) 434S 5.8 High 10527 IgG-pI-Iso2-SLCK-Iso(5) 434S 5.8 Low

Antibody variants were constructed with the variable region ofbevacizumab using molecular biology techniques as described above.Antibodies were expressed, purified, and characterized as describedabove. PK studies of the variant and control antibodies were carried outin the huFcRn mice as described above. The group mean averages of theserum concentrations as well as the half-lives obtained from fits of thedata for one of these variants (XENP10519—IgG-pI-Iso3-SL-434S-CK-Iso(5))are plotted in FIG. 32 and the half-lives for individual mice in FIG.33. This variant is also included in the correlation plot shown in FIG.34. The benefit of lower pI due to the CK-Iso(5) light chain is clearlyshown.

Example 8 Purifying Mixtures of Antibody Variants with ModifiedIsolectric Points

Substitutions that modify the antibody isoelectric point may beintroduced into one or more chains of an antibody variant to facilitateanalysis and purification. For instance, heterodimeric antibodies suchas those disclosed in US2011/0054151A1 can be purified by modifying theisolectric point of one chain, so that the multiple species presentafter expression and Protein A purification can be purified by methodsthat separate proteins based on differences in charge, such as ionexchange chromatography. An overview of the process using two differentheavy chains—one unmodified IgG1, and one with modified isolectricpoint, is shown in FIG. 38.

As an example, the heavy chain of bevacizumab was modified byintroducing substitutions to lower its isolectric point such that thedifference in charges between the three species produced whenWT-IgG1-HC, low-pI-HC, and WT-LC are transfected in 293E cells is largeenough to facilitate purification by anion exchange chromatography.Clones were created as described above, and transfection and initialpurification by Protein A chromatography is also as described above.Sequences of the three chains are listed in FIG. 39 as “Heavy chain 1 ofXENP10653”, “Heavy chain 2 of XENP10653”, and “Light chain ofXENP10653”. After Protein A purification, three species with nearlyidentical molecular weights, but different charges are obtained. Theseare the WT-IgG1-HC/WT-IgG1-HC homodimer (pI=8.12), WT-IgG1-HC/low-pI-HCheterodimer (pI=6.89), and low-pI-HC/low-pI-HC homodimer (pI=6.20). Themixture was loaded onto a GE HiTrap Q HP column in 20 mM Tris, pH 7.6and eluted with a step-wise gradient of NaCl consisting of 50 mM, 100mM, and finally 200 mM NaCl in the same Tris buffer. Elution wasmonitored by A280, and each fraction analyzed on Invitrogen pH 3-10 IEFgels with Novex running buffer and these results are shown in FIG. 40.WT-IgG1-HC/WT-IgG1-HC homodimer does not bind to the anion exchangecolumn at pH 7.6 and is thus present in the flowthrough and wash (lanes1-2). The desired heterodimer elutes with 50 mM NaCl (lane 3), while thelow-pI-HC/low-pI-HC homodimer binds tightest to the column and elutes at100 (lane 4) and 200 mM (lane 5) NaCl. Thus the desired heterodimervariant, which is difficult to purify by other means because of itssimilar molecular weight to the other two species, is easily purified bythe introduction of low pI substitutions into one chain. This method ofpurifying antibodies by engineering the isoelectric point of each chaincan be applied to methods of purifying various bispecific antibodyconstructs as outlined in FIG. 41 and FIG. 42. The method isparticularly useful when the desired species in the mixture has similarmolecular weight and other properties such that normal purificationtechniques are not capable of separating the desired species in highyield. Specific heterodimeric and/or bispecific constructs and sequenceswith isoelectric points engineered for easy purification are shown inTables 9 and 10, and FIG. 39, respectively.

TABLE 9 Heterodimeric and/or bispecific constructs with isoelectricpoints engineered for easy purification and list of isoelectric points.Calculated pI Low pI Hetero- High pI Protein Homodimer dimer HomodimerXENP10653 6.20 6.87 8.02 Anti-HER2 × anti-CD16 mAb-Fv 6.07 7.31 8.47Anti-CD19 × anti-CD16 mAb-Fv 5.84 6.63 8.21 Anti-CD19 × anti-CD32bmAb-Fv 6.23 6.74 7.80 Anti-CD40 × anti-CD32b mAb-Fv 6.54 7.46 8.22Anti-HER2 × anti-CD3 mAb-Fv 7.58 8.21 8.52 Anti-HER2 × anti-CD3 scFv-Fc7.31 8.31 8.69

TABLE 10 Heterodimeric and/or bispecific constructs with isoelectricpoints engineered for easy purification and list of charge state at pH7.4. Calculated charge state at pH 7.4 Low pI Hetero- High pI ProteinHomodimer dimer Homodimer XENP10653 −12.57 −3.59 +5.40 Anti-HER2 ×anti-CD16 mAb-Fv −16.67 −0.65 +15.37 Anti-CD19 × anti-CD16 mAb-Fv −22.68−6.66 +9.36 Anti-CD19 × anti-CD32b mAb-Fv −14.53 −5.59 +3.35 Anti-CD40 ×anti-CD32b mAb-Fv −8.51 +0.43 +9.37 Anti-HER2 × anti-CD3 mAb-Fv +1.25+9.32 +17.40 Anti-HER2 × anti-CD3 scFv-Fc −0.34 +6.68 +13.71

Example 9 Design of Non-Native Charge Substitutions to Alter pI

The pI of antibody constant chains were altered by engineeringsubstitutions in the constant domains. Reduced pI can be engineered bymaking substitutions of basic amino acids (K or R) to acidic amino acids(D or E), which result in the largest decrease in pI. Mutations of basicamino acids to neutral amino acids and neutral amino acids to acidicamino acids will also result in a decrease in pI. Conversely, increasedpI can be engineered by making substitutions of acidic amino acids (D orE) to basic amino acids (K or R), which result in the largest increasein pI. Mutations of acidic amino acids to neutral amino acids andneutral amino acids to basic amino acids will also result in a increasein pI. A list of amino acid pK values can be found in Table 1 ofBjellqvist et al., 1994, Electrophoresis 15:529-539.

In deciding which positions to mutate, the surrounding environment andnumber of contacts the WT amino acid makes with its neighbors was takeninto account such as to minimize the impact of a substitution or set ofsubstitutions on structure and/or function. The solvent accessibility orfraction exposed of each constant region position was calculated usingrelevant crystal structures. The results are shown in FIG. 43. Based onthis analysis, a number of substitutions were identified that reduce orincrease pI but are predicted to have minimal impact on the biophysicalproperties of the domains. Proof of concept results in the context ofbevacizumab are shown in FIGS. 44-47 (heavy chain) and FIGS. 48-51(light chain).

Calculation of protein pI was performed as follows. First, a count wastaken of the number of D, E, C, H, K, R, and Y amino acids as well asthe number of N- and C-termini present in the protein. Then, the pI wascalculated by identifying the pH for which the protein has an overallcharge of zero. This was done by calculating the net charge of theprotein at a number of test pH values. Test pH values were set in aniterative manner, stepping up from a low pH of 0 to a high pH of 14 byincrements of 0.001 until the charge of the protein reached or surpassedzero. Net charge of a protein at a given pH was calculated by thefollowing formula:

${q_{protein}({pH})} = {{\sum\limits_{{i = H},K,R,{Ntermini}}^{\;}\frac{N_{i}}{1 + 10^{{pH} - {pK}_{i}}}} - {\sum\limits_{{i = \; D},E,C,Y,{Ctermini}}^{\;}\frac{N_{i}}{1 + 10^{{pK}_{i} - {pH}}}}}$

where q_(protein)(pH) is the net charge on the protein at the given pH,is the number of amino acid i (or N- or C-termini) present in theprotein, and is the pK of amino acid i (or N- or C-termini).

Example 10 Isotypic Constant Region Variants

As described above, efforts can be made to minimize the risk thatsubstitutions that increase or decrease pI will elicit immunogenicity byutilizing the isotypic differences between the IgG subclasses (IgG1,IgG2, IgG3, and IgG4). A new set of novel isotypes was designed based onthis principal. If possible, pI-altering substitutions were accompaniedby isotypic substitutions proximal in sequence. In this way, epitopeswere extended to match a natural isotype. Such substitutions would thusmake up epitopes that are present in other human IgG isotypes, and thuswould be expected to be tolerized. These new variants are called ISO(−),ISO(+), and ISO(+RR). ISO(−) has reduced pI while ISO(+) and ISO(+RR)have increased pI. A sequence alignment showing the isotypic variationin IgG1-4 as well as the sequences of the new isotypic pI variants areshown in FIG. 52. The sequences of these new variants are also shown inFIG. 53-57. All possible combinations of pI lowering isotypic mutationsfrom IgG1, IgG2, IgG3, and IgG4 are shown in FIG. 58. All possiblecombinations of pI increasing isotypic mutations are shown in FIG. 59.

Example 11 Purifying Mixtures of Antibody Variants with ModifiedIsolectric Points

As mentioned previously, substitutions that modify the antibodyisoelectric point may be introduced into one or more chains of anantibody variant to facilitate analysis and purification. This isespecially useful when a preparation of antibody contains a mixture ofvery similar species as in the case of heterodimeric and/or bispecificconstructs that produce a mixture of hetero- and homodimers. In order todemonstrate purification of a nearly identical antibody heterodimerspecies from the corresponding homodimers, we constructed our isotypicpI variants in the context of the antibody bevacizumab. Variants wereconstructed by transfecting two different heavy chain DNAs (ISO(−),ISO(+), ISO(+RR), or IgG1(WT)) with the bevacizumab light chain.Variants were first purified by Protein A, and then loaded onto a GEHealthcare HiTrap SP HP cation exchange column in 50 mM MES (pH 6.0) andeluted with an NaCl gradient. Following elution, fractions from eachpeak were loaded onto a Lonza IsoGel IEF plate (pH range 7-11) foranalysis. Data are shown in FIGS. 60-63. As can be seen from the data,separation of the middle pI heterodimer is achieved in each case, withseparation improved when the heterodimer has a larger difference in pIfrom the homodimers.

Example 12 Design of Mixtures of Immunoglobulin Variants with ModifiedIsoelectric Points

This method of purifying antibodies by engineering the isoelectric pointof each chain can be applied to methods of purifying various bispecificantibody constructs. The method is particularly useful when the desiredspecies in the mixture has similar molecular weight and other propertiessuch that normal purification techniques are not capable of separatingthe desired species in high yield. Specific heterodimeric and/orbispecific constructs and sequences with isoelectric points engineeredfor easy purification are shown in FIG. 64.

1-54. (canceled)
 55. A composition comprising a heterodimeric proteincomprising: a) a first monomer comprising: i) a first variant heavychain constant region; ii) a first fusion partner; and b) a secondmonomer comprising: i) a second variant heavy chain constant region; ii)a second fusion partner; wherein the Fc region of said first and secondconstant regions comprise a set of amino acid substitutions selectedfrom the group consisting of the sets depicted in FIGS. 79, 80 and 82.56. A composition according to claim 55 wherein said heterodimericprotein has a structure selected from the group consisting of thestructures in FIG. 78A-78N.
 57. A composition according to claim 55wherein said first monomer comprises a third fusion partner.
 58. Acomposition according to claim 55 wherein said second monomer comprisesa fourth fusion partner.
 59. A composition according to claim 55 whereinsaid fusion partners are independently selected from the groupconsisting of an immunoglobulin component, a peptide, a cytokine, achemokine, an immune receptor and a blood factor.
 60. A compositionaccording to claim 59 wherein said immunoglobulin component is selectedfrom the group consisting of Fab, VH, VL, scFv, scFv2, dAb.
 61. Acomposition according to claim 59 wherein both fusion partners areimmunoglobulin components.
 62. A composition according to claim 55wherein the Fc domain of each monomer comprises chain comprises an aminoacid variant selected from the group consisting of 236A, 239D, 239E,332E, 332D, 239D/332E, 267D, 267E, 328F, 267E/328F, 236A/332E,239D/332E/330Y, 239D, 332E/330L, 236R, 328R, 236R/328R, 243L, 298A and299T.
 63. A composition according to claim 55 wherein the Fc domain ofeach monomer comprises chain comprises an amino acid variant selectedfrom the group consisting of 434A, 434S, 428L, 308F, 259I, 428L/434S,259I/308F, 436I/428L, 436I or V/434S, 436V/428L, 252Y, 252Y/254T/256Eand 259I/308F/428L.
 64. A nucleic acid encoding a first heavy chainaccording to claim
 55. 65. A nucleic acid encoding a second heavy chainaccording to claim
 55. 66. A host cell comprising the nucleic acid ofclaim 64 and the nucleic acid of claim
 65. 67. A method of making acomposition according to any previous claim comprising culturing a hostcell according to claim 66 under conditions whereby said composition isproduced.
 68. A method of treating an individual in need thereofcomprising administering the composition of claim 55.